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Amitriptyline
Molecular formula: C20H23N Molecular weight: 277.41 CAS Registry No.: 50-48-6, 549-18-8 (HCI) Merck Index: 511 LednicerNo.: 1 151,404

SAMPLE

Matrix: bile, blood, gastric contents, tissue, urine Sample preparation: Chop 5-g tissue and homogenize (Ultra Turrax T25) at 8500, 9500, 13500, 20500, and 24000 rpm for 1 min each. Add homogenate to 20 mL water. Dilute blood, urine, gastric contents, and bile four times with water. Mix 4 mL sample with 100 IxL 400 (xg/mL IS and 2 mL 500 mM NaOH, vortex briefly, add 4 mL heptane:isoamyl alcohol 98.5:1.5 and mix for 15 min (Spiramix 10, Denley, UK). Separate the organic layer, add 4 mL heptanerisoamyl alcohol 98.5:1.5 to extraction sample, mix. Combine the organic layers and extract them with 2 mL 50 mM sulfuric acid. Make the acid layer alkaline with 1 mL 1.0 M pH 9.0 carbonate/bicarbonate buffer and mix with 2 mL toluene:isoamyl alcohol 85:15 for 15 min. Evaporate the organic layer to dryness, reconstitute the residue in 100 |xL MeOH and inject a 20 |JLL aliquot.
HPLCVARIABLES

Guard column: 20 X 4.6 5 |xm Apex II ODS Column: 150 X 4.6 5 ^m Apex II OD Mobile phase: MeCN:pH 3 phosphate buffer:n-nonylamine 40-50:60:0.12 Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 4.48 Internal standard: doxepin (2.99) OTHER SUBSTANCES Extracted: nortriptyline
KEY WORDS

liver; lung; muscle; urine; pericardial fluid

REFERENCE Pounder,D.J.; Adams,E.; Fuke,C; Langford,A.M. Site to site variability of postmortem drug concentrations in liver and lung, J.Forensic ScL, 1996, 41, 927-932. SAMPLE

Matrix: blood Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 |xL plasma. Add 100 |xL 1 jjig/mL IS in MeOH, extract with 10 mL n-hexane. Shake for 30 min and centrifuge at 3000 g for 10 min. Cool in a dry ice-acetone bath. Add 200 |xL 0.3% phosphoric acid to upper organic layer. Shake for 10 min and centrifuge at 3000 g for 10 min. Separate the organic layer. Inject a 100 |xL aliquot of the acidic aqueous layer.
HPLC VARIABLES

Column: 250 X 4.6 5 jxm C18 Symmetry (Waters Millipore, USA)

Mobile phase: MeCN:67 mM potassium phosphate buffer adjusted to pH 3.0 with phosphoric acid 35:65 (After each chromatographic session wash the column with 200 mL MeCN:water 50:50.) Flow rate: 1.2 Injection volume: 100 Detector: UV 226, UV 254, UV 400
CHROMATOGRAM

Retention time: 11.53 Internal standard: clovoxamine (6.5) Limit of quantitation: 5 ng/mL (UV 226, UV 400); 7 ng/mL (UV 254)
OTHER SUBSTANCES

Extracted: metabolites, clomipramine, desipramine, fluoxetine, imipramine maprotiline, nortriptyline Simultaneous: amineptine, carbamazepine, chlordiazepoxide, chlorpromazine, clonazepam, clorazepate, clozapine, cyamemazine, desmethylmaproptiline, desmethylvenlafaxine, doxepin, flunitrazepam, fluvoxamine, haloperidol, lorazepam, loxapine, mianserine, sulpiride, trimipramine, venlafaxine, viloxazine, zolpidem, zopiclone Noninterfering: diazepam, valproic acid Interfering: levomepromazine
KEY WORDS

plasma
REFERENCE Aymard,G.; Livi,R; Pham,Y.T.; Diquet,B. Sensitive and rapid method for the simultaneous quantification of five antidepressants with their respective metabolites in plasma using high-performance liquid chromatography with diode-array detection, J.Chromatogr.B, 1997, 700, 183-189. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL 30 mg Oasis HLB SPE cartridge with 1 mL MeOH and 1 mL water. Acidify (?) mL serum with 20 jxL phosphoric acid, vortex for 5 s, add to the SPE cartridge, wash with 1 mL MeOH :water 5:95, elute with 1 mL MeOH. Evaporate the eluate to dryness at 40 under a stream of nitrogen. Reconstitute the residue with 200 uL MeOH:20 mM pH 7 phosphate buffer 20:80, inject a 20 u-L aliquot.
HPLC VARIABLES

Guard column: 20 X 3.9 Sentry Column: 150 X 3.9 5 |xm Symmetry C18 (Waters) Mobile phase: MeOH:20 mM pH 7 potassium phosphate 70:30 Column temperature: 35 Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 21 Internal standard: nordoxepin (4.9)

OTHER SUBSTANCES

Simultaneous: metabolites, doxepin, nortriptyline

KEY WORDS

pig; serum; SPE

REFERENCE Cheng,Y.-R; Phillips,D.J.; Neue,U.; Bean,L. Solid-phase extraction for the determination of tricyclic antidepressants in serum using a novel polymeric extraction sorbent, J.Liq.Chromatogr.Rel.Technol., 1997, 20, 2461-2473. SAMPLE

Matrix: blood, gastric contents, tissue Sample preparation: Blood. Mix 200 |xL blood with 200 |xL pH 11 borate buffer and 40 JJLL aqueous trimipramine solution. Extract with 800 JJLL hexane:2-butanol 98:2, centrifuge at 740 g for 10 min, evaporate the organic phase under vacuum at 40 (Buchler Vortex Evaporator, USA). Dissolve the residue in 100 |xL mobile phase, inject an aliquot. Tissue, gastric contents. Homogenize tissue in pH 11 borate buffer to a final concentration 200 mg tissue/mL homogenate (Ultraturrax T5 homogenizer, IKA, Germany), dilute gastric contents 1:9 with water. Mix 200 |JLL tissue homogenate or diluted gastric contents with 40 (xL aqueous trimipramine solution. Extract with 800 |xL hexane:2-butanol 98:2, centrifuge at 740 g for 10 min, evaporate the organic phase under vacuum at 40 (Buchler Vortex Evaporator, USA). Dissolve the residue in 100 jxL mobile phase, inject a 20 (xL aliquot. (Buffer was saturated aqueous sodium tetraborate adjusted to pH 11 with 6 M NaOH.)
HPLC VARIABLES

Column: 250 X 4.6 5 p,m Supelcosil LC-Si Mobile phase: MeCN:25 mM ammonium acetate 90:10 Flow rate: 3 Injection volume: 20 Detector: UV 230
CHROMATOGRAM

Internal standard: trimipramine maleate Limit of detection: 40 nmol/kg (blood), 200 nmol/kg (tissue)
OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

whole blood; bone marrow; kidney; liver; lung; muscle; pharmacokinetics; pig; vitreous humor
REFERENCE Hilberg,T.; Ripel,.; Smith,A.J.; Slordal,L.; Morland,J.; Bjorneboe,A. Postmortem amitriptyline pharmacokinetics in pigs after oral and intravenous routes of administration, J.Forensic ScL, 1998, 43, 380387. SAMPLE

Matrix: blood, microsomal incubations Sample preparation: Vortex 1 mL plasma or microsomal incubation with 200 |JLL 1 jig/mL desipramine and 100 JULL 5 M NaOH for 10 s, add 5 mL butan-l-ol:hexane 2:98, vortex for 1 min, centrifuge at 2000 g and 4 for 5 min, evaporate the organic phase to dryness at 40 using a vacuum vortex evaporator, reconstitute the residue in 200 |xL mobile phase,
inject a 50 JJIL aliquot. HPLC VARIABLES

Column: 5 |xm Nova-Pak C18 Mobile phase: MeCN:buffer 30:70 (Buffer was water containing 1% triethylamine, adjusted to pH 3 with orthophosphoric acid.) Flow rate: 2 Injection volume: 50

Detector: UV 240
CHROMATOGRAM

Retention time: 10.5 Internal standard: desipramine (6.3) Limit of quantitation: 2 ng/mL OTHER SUBSTANCES Extracted: nortriptyline Simultaneous: ketoconazole Noninterfering: diazepam, furafylline, hydroxyamitriptyline, hydroxynortriptyline, quinidine, mephenytoin, triacetyloleandomycin
KEYWORDS

human; liver; rat; plasma

REFERENCE Ghahramani,R; Lennard,M.S. Quantitative analysis of amitriptyline and nortriptyline in human plasma and liver microsomal preparations by high-performance liquid chromatography, J.Chromatogr.B, 1996, 685, 307-313. SAMPLE

Matrix: blood, tissue, urine Sample preparation: Serum, urine. 500 |ULL Serum or urine + 100 |xL 2 |xg/mL diazepam + 200 |xL 20% sodium carbonate + 500 |xL water + 3 mL n-hexane:isoamyl alcohol 98.5: 1.5, mix for 2 min, centrifuge at 1200 g for 5 min. Remove the organic phase and evaporate it under a gentle stream of nitrogen at about 40. Dissolve the residue in 100 jxL mobile phase, inject a 10 jxL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 jxg/mL diazepam, centrifuge at 15 000 g for 10 min. Add 500 jxL 20% sodium carbonate and 4 mL n-hexane:isoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic phase and evaporate it under a gentle stream of nitrogen at about 40. Dissolve the residue in 100 JJLL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 2 ^m TSK gel Super-Octyl (A) or 100 X 4.6 5 jxm Hypersil MOS-C8 (B) Mobile phase: MeOH:20 mM pH 7 KH2PO4 60:40 Flow rate: 0.6 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 16.2 (A), 28.5 (B) Internal standard: diazepam (4.4, A) Limit of quantitation: 50 ng/mL (serum, urine) (A), 500 ng/mL (tissue) (A)
OTHER SUBSTANCES

Extracted: amoxapine, clomipramine, desipramine, dothiepin, doxepin, imipramine, maprotiline, melitracen, mianserin, nortriptyline Noninterfering: barbital, carbamazepine, ethosuximide, hexobarbital, lofepramine, pentobarbital, phenobarbital, phenytoin, primidone, sulphide, trimethadione, trimipramine
KEYWORDS

serum; brain; liver

REFERENCE Tanaka,E.; Terada,M.; Nakamura,T.; Misawa,S.; Wakasugi,C. Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 |xm porous microspherical silica gel, J.Chromatogr.B, 1997, 692, 405-412. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 fxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLCVARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 206.4 CHROMATOGRAM Retention time: 15.878 KEYWORDS whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: serum Sample preparation: 1 mL Serum + 500 |xL 750 mM pH 10 sodium bicarbonate/carbonate buffer + 50 jxL IS in EtOHrwater 50:50 + 8 mL heptanerisoamyl alcohol 98:2, shake at 250 cycles/min for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Remove the organic layer and add it to 150 |xL 22 mM pH 2.5 KH^PCVphosphoric acid buffer, shake at 250 cycles/min for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Discard the organic layer, inject a 65 |xL aliquot of the aqueous layer.
HPLC VARIABLES

Column: 250 X 4.6 Supelco C18 Mobile phase: MeCN:buffer 45:55 (Buffer was 44 mM KH2PO4 containing 1.5 mIVL triethylamine, adjusted to pH 2.5 with phosphoric acid.) Flow rate: 1.5 Injection volume: 65 Detector: UV 240

CHROMATOGRAM Retention time: 11.5 Internal standard: l-(3-(dimethylamino)propyl)-l-(p-chlorophenyl)-l,3-dihydroisobenzofuran-5-carbonitrile (LU 10-202) (Lundbeck, Copenhagen) (8.33)
OTHER SUBSTANCES

Extracted: citalopram, nortriptyline Simultaneous: chlorprothixene, clomipramine, clozapine, flupenthixol, haloperidol, levomepromazine, perphenazine, zuclopenthixol Noninterfering: benzodiazepines Interfering: didesmethylclomipramine, levomepromazine
KEY WORDS

serum
REFERENCE Olesen,O.V.; Linnet,K. Simplified high-performance liquid chromatographic method for the determination of citalopram and desmethylcitalopram in serum without interference from commonly used psychotropic drugs and their metabolites, J.Chromatogr.B, 1996, 675, 83-88. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4 ODS (Hitachi) Mobile phase: MeCN:50 mM phosphoric acid 50:50 containing 300 mM KCl Column temperature: 55 Flow rate: 0.6 Injection volume: 20 Detector: UV 239
OTHER SUBSTANCES

Also analyzed: chlorpromazine, clomipramine, promazine, promethazine, thymol

REFERENCE Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K.; Miyazaki,K. A general approach for the prediction of the intestinal absorption of drugs: regression analysis using the physicochemical properties and drug-membrane electrostatic interactions, J.Pharm.Sci., 1998, 87, 960-966.

Amlexanox
Molecular formula: C16H14N2O4 Molecular weight: 298.30 CAS Registry No.: 68302-57-8 Merck Index: 515

SAMPLE

Matrix: tissue Sample preparation: Cut eye tissue in small pieces, add 5 mL MeoH, shake for 30 min, let stand overnight, centrifuge at 3000 rpm for 10 min. Remove 4 mL of the supernatant and evaporate it to dryness under vacuum, reconstitute the residue in 500 fxL mobile phase, inject a 20 (xL aliquot.
HPLC VARIABLES

Column: Schim-pack C18 Mobile phase: MeCN:50 mM pH 8.0 NaH2PO4 25:73 Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: F ex 350 em 402
CHROMATOGRAM

Internal standard: 4-methylumbelliferone Limit of detection: < 4000 ng/g

KEYWORDS

rat; eye
REFERENCE Rankov,G.; Sasaki,K; Fukuda,M. Pharmacodynamics of Amlexanox (AA-673) in normal and anaphylactic rat conjunctiva and its effect on histamine concentration, Ophthalmic Res., 1990, 22, 359-364.

Amlodipine
Molecular formula: C20H25CIN2O5 Molecular weight: 408.88 CAS Registry No.: 88150-42-9, 111470-99-6 (besylate), 88150-47-4 (maleate) Merck Index: 516 Lednicer No.: 4 108

SAMPLE

Matrix: blood Sample preparation: Mix 5.0 mL plasma with 250 ng chloroamlodipine, add 250 fiL 1 M sodium hydroxide and 5 mL chloroform, shake for 10 min, centrifuge at 3500 g. Dry the organic layer in a vacuum centrifuge, wash the tube twice with 500 jxL chloroform and dry again. Dissolve the extract in 70 (xL MeOH:pH 4.5 acetic buffer 60:40, inject a 50 |xL aliquot. (For added sensitivity each eluting enantiomer may be trapped separately on 20 X 4.6 5 |xm Supelcosil LC8 columns and eluted from these columns with MeCN: 10 mM pH 4.5 acetate buffer 45:55 and chromatographed on 150 X 4.6 4 |xm Symmetry C 8 columns.)
HPLC VARIABLES

Column: 150 X 4 Chiral AGP (ChromTech, Haegerstern, Sweden) Mobile phase: 1-Propanol:10 mM pH 4.5 acetate buffer 1:99 Column temperature: 30 Flow rate: 0.9 Injection volume: 50 Detector: UV 240
CHROMATOGRAM

Retention time: 11. 5 (R-(+)-), 15.4 (S-(-)-) Internal standard: chloroamlodipine (29.2 (R-(+)), 36.6 (S-(-))
KEYWORDS

plasma; chiral; pharmacokinetics

REFERENCE Luksa,J.; Josic,D.; Kremser,M.; Kopitar,Z.; Milutinovic,S. Pharmacokinetic behaviour of R-(+)- and S(-)-amlodipine after single enantiomer administration, J.Chromatogr.B, 1997, 703, 185-193. SAMPLE

Matrix: blood Sample preparation: 5 mL Plasma + 250 ng chloramlodipine + 250 |xL 1 M NaOH + 5 mL chloroform, shake for 10 min, centrifuge at 3500 g. Evaporate the organic layer to dryness in a vacuum centrifuge, add 500 |xL chloroform to the residue, evaporate to dryness, add 500 jxL chloroform to the residue, evaporate to dryness, reconstitute with 70 IxL MeOH:pH 4.5 acetate buffer 60:40, inject a 50 |xL aliquot onto column A and elute to waste with mobile phase A, collect each enantiomer on SEPARATE columns B. After 40 min elute the column B containing R-amlodipine with mobile phase B onto column C, monitor the effluent from column C, later elute the column B containing S-amlodipine with mobile phase B onto column C, monitor the effluent from column C.
HPLC VARIABLES

Column: A 150 X 4 Chiral AGP (ChromTech); B 20 X 4.6 Supelcosil LC-8; C 150 X 4.6 Symmetry C8 (Waters) Mobile phase: A n-Propanol:10 mM pH 4.5 acetate buffer 1:99; B MeCN:10 mM pH 4.5 acetate buffer 45:55

Column temperature: 30 (column C only) Flow rate: 0.9 Injection volume: 50 Detector: UV 240
CHROMATOGRAM

Retention time: 42.9 (R-(+)), 58.3 (S-(-)) [after start of analysis] Internal standard: chloramlodipine (Lek) Limit of detection: <5 ng/mL
KEYWORDS

chiral; plasma; column-switching

REFERENCE Luksa,J.; Josic,D.; Podobnik,B-; Furlan,B; Kremser,M. Semi-preparative chromatographic purification of the enantiomers of S-(-)-amlodipine and R-(+)-amlodipine, J.Chromatogr.B, 1997, 693, 367-375. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |JLL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JJLL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |im Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 15.093 KEYWORDS

whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: solutions Sample preparation: Inject an 800 |xL aliquot of a 1.3 mg/mL solution.
HPLC VARIABLES

Column: 150 X 10 Chiral AGP (ChromTech, Sweden)

Mobile phase: n-Propanol:10 mM pH 4.5 acetate buffer 1:99 Flow rate: 4 Injection volume: 800 Detector: UV 240
CHROMATOGRAM

Retention time: 35 (R-(+)), 48 (S-(-))

KEYWORDS

chiral; semi-preparative
REFERENCE Luksa,J.; Josic,D.; Podobnik,B-; Furlan,B-; Kremser,M. Semi-preparative chromatographic purification of the enantiomers of S-(-)-amlodipine and R-(+)-amlodipine, J.Chromatogr.B, 1997, 693, 367-375.

Amobarbital
Molecular formula: C11H18N2O3 Molecular weight: 226.28 CAS Registry No.: 57-43-2, 64-43-7 (sodium salt) Merck Index: 607 Lednicer No.: 1 268

SAMPLE

Matrix: blood Sample preparation: 200 jxL Serum + 200 jxL 50 fjig/mL hexobarbital in MeCN + 25 jxL glacial acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 JJLL aliquot of the supernatant.
HPLC VARIABLES

Column: ixBondapak C18 Mobile phase: Gradient. MeCN:7.5 g/L NaH2PO4 adjusted to pH 3.2 with phosphoric acid 5:95 to 22:78 over 24 min, to 45:55 over 10 min, maintain at 45:55 for 5 min. Re-equilibrate with 5:95 for 5 min. Column temperature: 50 Flow rate: 3 Injection volume: 30-100 Detector: UV 210
CHROMATOGRAM

Retention time: 23.0 Internal standard: hexobarbital (20.6) Limit of detection: 200-2000 ng/mL
OTHER SUBSTANCES

Extracted: acetaminophen, butabarbital, butalbital, chlordiazepoxide, diazepam, ethchlorvynol, flurazepam, glutethimide, methaqualone, methyprylon, nitrazepam, pentobarbital, phenobarbital, phenytoin, primidone, salicylic acid, secobarbital, theophylline Simultaneous: amitriptyline, caffeine, clomipramine, codeine, desipramine, ethotoin, imipramine, lidocaine, mesantoin, methsuximide, nirvanol, nortriptyline, oxazepam, procainamide, phenylpropanolamine, propranolol, quinidine
KEY WORDS

serum
REFERENCE Kabra,P.M.; Stafford,B.E.; Marton,L.J. Rapid method for screening toxic drugs in serum with liquid chromatography, JAnalToxicol, 1981, 5, 177-182. SAMPLE

Matrix: blood Sample preparation: 100 |JLL Serum + 100 |xL buffer + 1.5 mL IS in 5% isopropanol in chloroform, vortex for 30 s, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of air at room temperature, reconstitute the residue in 100 fxL mobile phase, inject a 6-10 |xL aliquot. (Buffer was 13.6 g KH2PO4 in 90 mL water, pH adjusted to 6.8 with about 3 mL 10 M NaOH, made up to 100 mL.)
HPLC VARIABLES

Guard column: 20 X 4.6 Supelguard LC-I (Supelco) Column: 250 X 4.6 5 jxm Supelcosil LC-I (Supelco)

Mobile phase: MeOH:MeCN:buffer 17.5:17.5:65 (Buffer was 2.72 g KH2PO4 in 1.9 L water, pH adjusted to 6.3 with about 2 mL 1 M NaOH, made up to 2 L.) Flow rate: 2 Injection volume: 6-10 Detector: UV 204
CHROMATOGRAM

Retention time: 6.05 Internal standard: 5-ethyl-5-p-tolybarbituric acid (tolybarb) (4.80)

OTHER SUBSTANCES

Extracted: acetaminophen, barbital, caffeine, carbamazepine, chloramphenicol, ethosuximide, mephobarbital, methsuximide, pentobarbital, phenobarbital, phenytoin, primidone, secobarbital, theophylline, thiopental Also analyzed: acetanilide, N-acetylcysteine, N-acetylprocainamide, ampicillin, aspirin, butabarbital, butalbital, chlorpropamide, cimetidine, codeine, cyheptamide, diazoxide, diflunisal, diphylline, disopyramide, ethchlorvynol, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibuprofen, indomethacin, ketoprofen, mefenamic acid, mephenytoin, methaqualone, methsuximide, methyl salicylate, methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, phenacetin, phensuximide, phenylbutazone, procainamide, salicylamide, salicylic acid, sulfamethoxazole, sulindac, tolmetin, trimethoprim, vancomycin Noninterfering: amikacin, gentamicin, meprobamate, netilmicin, quinidine, tetracycline, tobramycin, valproic acid
KEYWORDS

serum
REFERENCE Meatherall,R-; Ford,D. Isocratic liquid chromatographic determination of theophylline, acetaminophen, chloramphenicol, caffeine, anticonvulsants, and barbiturates in serum, Ther.Drug MoniL, 1988, 10, 101-115. SAMPLE

Matrix: blood Sample preparation: Prepare an SPE cartridge by plugging the end of a 1 mL disposable pipette tip with glass wool and adding about 100 mg Chromosorb P/NAW. Add 50 |xL plasma then 50 jxL 10 jxg/mL tolylphenobarbital in 200 mM HCl to the SPE cartridge, let stand for 2 min, elute with 1 mL chloroformdsopropanol 6:1. Evaporate the eluate to dryness under a stream of nitrogen at 30, reconstitute the residue in 100 (xL mobile phase, inject a 15 JJLL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 fim Supelcosil-LC-8 Mobile phase: MeCNrwater 20:80 Flow rate: 3.3 Injection volume: 15 Detector: UV 208
CHROMATOGRAM

Retention time: 9.64 Internal standard: tolylphenobarbital (7.57) Limit of detection: 50-100 ng/mL
OTHER SUBSTANCES

Extracted: theophylline, caffeine, barbital, ethosuximide, primidone, carbamazepinediol, phenacemide, methyprylon, nirvanol, phenobarbital, chloramphenicol, butabarbital, carbamazepine epoxide, mephenytoin, pentobarbital, carbamazepine, glutethimide, phenytoin, secobarbital, methaqualone

Noninterfering: acetaminophen, amikacin, amitriptyline, clonazepam, cyclosporine, desipramine, diazepam, digoxin, disopyramide, gentamicin, imipramine, lidocaine, methotrexate, N-acetylprocainamide, netilmicin, nortriptyline, procainamide, quinidine, salicylic acid, sulfamethoxazole, tobramycin, trimethoprim, valproic acid, p-hydroxyphenobarbital, vancomycin
KEY WORDS

plasma; SPE
REFERENCE Svinarov,D.A.; Dotchev,D.C. Simultaneous liquid-chromatographic determination of some bronchodilators, anticonvulsants, chloramphenicol, and hypnotic agents, with Chromosorb P columns used for sample preparation, Clin.Chem., 1989, 35, 1615-1618. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 16.617
KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: bulk, formulations Sample preparation: Bulk quantities. Dissolve 25 mg bulk quantities in 25 mL MeCN, dilute 1:10 with MeCN, inject an aliquot. Tablets. Crush tablet, add 46 mg to 10 mL MeCN, shake for 24 h, dilute 1 mL clear supernatant 1:10 with MeCN, inject an aliquot. Capsules. Powder capsule, add 35 mg to 1 mL 10% sulfuric acid, shake, make up to 10 mL with MeCN, shake for 24 h, dilute 1 mL clear supernatant 1:10 with MeCN, inject an aliquot.

HPLC VARIABLES

Guard column: 20 X 2 37-53 |xm Whatman pellicular ODS Column: 250 X 4.6 5 ^m Econosphere C18 (Alltech) Mobile phase: MeCN:water 30:70 Flow rate: 1.2 Detector: UV 198 CHROMATOGRAM Retention time: 12.5 OTHER SUBSTANCES Simultaneous: impurities
KEY WORDS

tablets; capsules
REFERENCE Soine,W.H.; Graham,R.M.; Soine,P.J. Identification of 5-ethyl-5-(2-methylbutyl)barbituric acid as an impurity of manufacture in amobarbital, J.Pharm.ScL, 1992, 81, 362-364. SAMPLE

Matrix: saliva Sample preparation: Saliva sample obtained by chewing on 100 sq. cm Parafilm, centrifuge at 3000 g for 5 min. Remove a 1 mL aliquot of the supernatant and add it to 100 jxL 5 |xg/mL hexobarbital solution, add the mixture to a 3 mL octadecyl Baker-10 SPE cartridge, wash with three 3 mL portions of water, elute with 800 jxL MeOH, inject a 50
JULL aliquot of the eluate. HPLC VARIABLES

Guard column: 30 X 4 5 |xm Develosil ODS-5 (Nomura) Column: 150 X 4 5 |xm Develosil ODS-5 (Nomura) Mobile phase: MeOH:water 50:50 Flow rate: 0.8 Injection volume: 50 Detector: UV 240 following a 150 X 3 sulfonated hollow-fiber membrane reactor (Dionex AFS-2) which was surrounded by 50 mM pH 10.2 ammonium hydroxide solution
CHROMATOGRAM

Retention time: 14 Internal standard: hexobarbital (10) Limit of detection: 0.5-2.5 ng OTHER SUBSTANCES Simultaneous: barbital, phenobarbital
KEY WORDS

SPE; post-column reaction

REFERENCE Haginaka,J.; Wakai,J. Liquid chromatographic determination of barbiturates using a hollow-fibre membrane for postcolumn pH modification, J.Chromatogr., 1987, 390, 421-428. SAMPLE

Matrix: solutions Sample preparation: Mix 50 |xL of a 20-200 |xg/mL solution in acetone with 50 |JLL of a 0.4-1.6 mg/mL solution of 2-bromo-2'-acetonaphthone in acetone, add 5-10 mg cesium carbonate, heat at 30 for 30 min, add 50 |JLL glacial acetic acid, mix, inject an aliquot.

HPLC VARIABLES

Column: 300 x 4 jjiBondapak C18 Mobile phase: MeOHrwater 80:20 Flow rate: 2 Detector: UV 249 CHROMATOGRAM Retention time: 8 Limit of detection: 1 ng
OTHER SUBSTANCES

Simultaneous: barbital, butobarbital, heptobarbital, hexobarbital, mephobarbital, pentobarbital, phenobarbital, secobarbital

KEYWORDS

derivatization
REFERENCE Hulshoff,A.; Roseboom,H.; Renema,J. Improved detectability of barbiturates in high-performance liquid chromatography by pre-column labelling and ultraviolet detection, J.Chromatogr., 1979, 186, 535541. SAMPLE

Matrix: solutions Sample preparation: Evaporate a solution in water, MeOH, or diethyl ether to dryness, add a 3-fold molar excess of triethylamine, add 0.5-3 mL MeCN, add a 3-fold molar excess of N-chloromethyl-4-nitrophthalimide, heat at 60 for 1 h, inject an aliquot. (Preparation of N-chloromethyl-4-nitrophthalimide is as follows. Suspend 130 g 4-nitrophthalimide in 80 mL 40% formaldehyde solution, add 200 mL water, reflux for 4 h, filter while hot, N(hydroxymethyl-4-nitrophthalimide crystallizes on cooling (cf. J. Am. Chem. Soc. 1922, 44, 817). Mix a suspension of 2.26 g N-hydroxymethyl-4-nitrophthalimide in 10-15 mL ether with a suspension of 2.1 g phosphorus pentachloride in 10-15 mL ether, after 10 min heat on a water bath, cool in an ice-salt mixture, add ice-water dropwise with shaking, filter to obtain N-chloromethyl-4-nitrophthalimide, dry under vacuum (cf. Chem. Ber. 1959, 9, 1258).)
HPLC VARIABLES

Column: 7 |xm LiChrosorb RP8 Mobile phase: MeCN:water 60:40 Flow rate: 1.5 Detector: UV 254 CHROMATOGRAM Retention time: 7.2 Limit of detection: 4 ng
OTHER SUBSTANCES

Extracted: secobarbital Simultaneous: cyclobarbital, methylphenobarbital, phenobarbital

KEYWORDS

derivatization
REFERENCE Lindner,W.; Santi,W. N-chloromethylphthalimides as derivatization reagents for high-performance liquid chromatography, J.Chromatogr., 1979, 176, 55-64.

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4 OmniPac PAX-500 (Dionex) Mobile phase: Gradient. A was MeCN:5 mM sodium carbonate 9:81. B was MeCN:20 mM sodium carbonate 20:80. A:B from 100:0 to 0:100 over 10 min. Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: 10
OTHER SUBSTANCES

Simultaneous: allobarbital, barbital, barbituric acid, butabarbital, mephobarbital, methabarbital, methohexital, phenobarbital, phenytoin, secobarbital, thiamylal
REFERENCE SlingsbyR.W.; Rey,M. Determination of Pharmaceuticals by multi-phase chromatography: Combined reversed phase and ion exchange in one column, J.Liq.Chromatogr., 1990, 13, 107-134. SAMPLE

Matrix: solutions Sample preparation: Dissolve in mobile phase to a concentration of 50 jxg/mL.

HPLC VARIABLES

Column: 250 X 4 p-cyclodextrin polymer-coated silica (Chromatographia 1993, 36, 373) Mobile phase: MeOH:water 50:50 Flow rate: 0.6 Injection volume: 20 Detector: UV 240 CHROMATOGRAM Retention time: k' 1.95
OTHER SUBSTANCES

Simultaneous: aprobarbital, pentobarbital, butabarbital, butalbital, secobarbital, thiopental, phenobarbital

REFERENCE Forgacs,E.; Cserhati,T. Retention behaviour of barbituric acid derivatives on a p-cyclodextrin polymercoated silicon column, J.ChromatogrA, 1994, 668, 395-402. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210

OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine

REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994, 18, 233-242. SAMPLE

Matrix: solutions Sample preparation: Dissolve in mobile phase at a concentration of 100 |xg/mL, inject a
5 JULL aliquot.

HPLC VARIABLES

Column: 300 X 2 ixBondapak C18 Mobile phase: MeCN:water 30:70 adjusted to pH 3.0 with formic acid Flow rate: 0.27 Injection volume: 5 Detector: MS, VG TRIO 2000 single quadrupole MS with EI or CI or UV 270 CHROMATOGRAM Retention time: 14
OTHER SUBSTANCES

Extracted: butethal, butabarbital, talbutal, butalbital, pentobarbital

KEYWORDS

mass spectra given

REFERENCE Ryan,T.W. Identification of barbiturates using high performance liquid chromatography-particle beam EI/CI mass spectrometry, J.Liq.Chromatogr., 1994, 27, 867-881. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acidrbufifer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 5.94 (A), 5.59 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, pheno-

barbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119. SAMPLE

Matrix: urine Sample preparation: 200 JJLL Urine + 1 mL saturated ammonium sulfate, extract with 2 mL ethyl acetate. Remove the organic layer and evaporate it to dryness under vacuum, reconstitute the residue in 200 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jxm Alltech C18 Mobile phase: MeCN:25 mM pH 6.5 sodium phosphate buffer 20:80 Flow rate: 1.4 Injection volume: 20 Detector: UV 198
CHROMATOGRAM Retention time: 41.5 OTHER SUBSTANCES

Extracted: metabolites, conjugates

REFERENCE Soine,W.H.; Soine,P.J.; Overton,B.W.; Garrettson,L.K. Product enantioselectivity in the N-glucosylation of amobarbital, Z>ra# Metab.Dispos., 1986, 14, 619-621.

Amodiaquin
Molecular formula: C20H22CIN3O Molecular weight: 355.87 CAS Registry No.: 86-42-0, 6398-98-7 (dihydrochloride dihydrate) Merck Index: 609 Lednicer No.: 4 140

SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol:n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 JXL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 fim NovaPack C18 Mobile phase: MeOH.THF.buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 225
CHROMATOGRAM

Retention time: 4.62 Limit of detection: <120 ng/mL

KEY WORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol;

aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Rintz,P.; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262.

Amosulalol
Molecular formula: C18H24N2O5S Molecular weight: 380.47 CAS Registry No.: 85320-68-9 Merck Index: 614

SAMPLE

Matrix: blood Sample preparation: Add 1 mL saturated sodium bicarbonate solution and 100 (xL water to 1.5 mL plasma, extract with 5 mL ethyl acetate. Remove the organic layer and add it to 2.5 mL 400 mM HCl. Shake, centrifuge, and discard the organic layer. Add 2 mL saturated sodium bicarbonate solution to the aqueous layer and extract again with 5 mL ethyl acetate. Evaporate the organic layer to dryness under reduced pressure, reconstitute the residue with 50 jxL 100 mM sodium bicarbonate. Add 100 jxL 5 mg/mL dansyl chloride in acetone. Heat at 35 for 90 min. Add 5 mL distilled water and extract with 5 mL diethyl ether. Evaporate the organic layer to dryness at 45, reconstitute the residue with 60 |xL mobile phase. Inject a 20-50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 Nucleosil SI100-5 (Chemco, Japan) Mobile phase: MeOH:benzene 1:100 (Caution! Benzene is a carcinogen!) Injection volume: 20-50 Detector: F ex 352 em 500
KEY WORDS

amosulalol is IS; plasma; human; rat; dog; normal phase

REFERENCE Matsushima,H-; Kamimura,H.; Soeishi,Y.; Watanabe,T.; Higuchi,S.; Tsunoo,M. Pharmacokinetics and plasma protein binding of tamsulosin hydrochloride in rats, dogs, and humans, Drug Metab.Dispos., 1998, 26, 240-245. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 2 mL 0.5 |xg/mL IS in water + 0.5 g sodium bicarbonate + 4 mL ethyl acetate, extract, centrifuge. Remove the organic layer and evaporate it to dryness under reduced pressure, reconstitute the residue in 100 |xL 3 mg/mL sodium bicarbonate in water and 200 |xL 500 |xg/mL 5-di-n-butylaminonaphthalene-l-sulfonyl chloride (Bans-Cl) in acetone, heat at 45 for 90 min, cool, add 4 mL diethyl ether, wash mixture with 3 mL water for 10 s. Remove the organic layer and evaporate it to dryness at 40-50, reconstitute the residue in 100 jxL benzene (CAUTION! Benzene is a carcinogen!), inject a 5-10 |xL aliquot. (Derivatization can also be performed with 5-dimethylaminonaphthalene-1-sulfonyl chloride (Dans-Cl), F ex 250 em 505, retention time of derivative 4.1 min.)
HPLC VARIABLES

Column: 150 X 4 5 |mm LiChrosorb SI-60 Mobile phase: Benzene.MeOH 50:1 (CAUTION! Benzene is a carcinogen) Flow rate: 2 Injection volume: 5-10 Detector: F ex 356 em 500 CHROMATOGRAM Retention time: 2.9

Internal standard: 5-[l-hydroxy-2-[2-(o-methoxyphenoxy)ethylamino]ethyl]-2-methoxybenzenesulfonamide (4.0) Limit of detection: 20 ng/mL

KEYWORDS

plasma; normal phase; derivatization; dog; pharmacokinetics

REFERENCE Kamimura,H.; Sasaki,H.; Kawamura,S. Determination of the a,(3-adrenoceptor blocker YM-09538 in plasma by high-performance liquid chromatography with fluorescence detection, J.Chromatogr., 1981, 225, 115-121.

Amoxapine
Molecular formula: C17H16CIN3O Molecular weight: 313.79 CAS Registry No.: 14028-44-5 Merck Index: 616 Led nicer No.: 2 428

SAMPLE

Matrix: blood Sample preparation: 500 |JLL Serum + 50 (JLL 100 mM HCl + 400 JJLL 1 M NaOH + 6 mL ethyl acetate, shake for 15 min, centrifuge at 4000 g for 10 min. Transfer top organic layer to another tube and re-extract the analyte with 3 mL 50 mM HCl. Evaporate organic layer in water bath under nitrogen at 45. Dissolve the residue in 120 |xL mobile phase, inject a 30 |ULL aliquot.
HPLC VARIABLES

Guard column: Perisorb RP-8 (Upchurch) Column: 100 X 4.6 3 u.m Supelco C8-DB Mobile phase: MeCN:MeOH:buffer 20:18:62 (Buffer was 50 mM sodium monobasic phosphate, contains 2.5 mL/L triethylamine, pH adjusted to 2.7 with phosphoric acid.) Flow rate: 1.3 Injection volume: 30 Detector: UV 230 CHROMATOGRAM Retention time: 14.3 Internal standard: amoxapine
OTHER SUBSTANCES

Extracted: metabolites, clozapine Simultaneous: amitryptiline, atenolol, bupropion, cogentin, desipramine, diazepam, doxepin, fluoxetine, haloperidol, imipramine, loxapine, medazepam, nortryptiline, oxazepam, paroxetine, phenytoin, propranolol, sertraline, thiothixene, trazadone, trifluoperazine, valproic acid, verapamil Interfering: carbamazepine, desmethylsertraline
KEY WORDS

serum; amoxapine is IS
REFERENCE Hariharan,U.; Hariharan,M.; Naickar,J.S.; Tandon,R. Determination of clozapine and its two major metabolites in human serum by liquid chromatography using ultraviolet detection, J.Liq. Chromatogr.Rel.TechnoL, 1996, 19, 2409-2417. SAMPLE

Matrix: blood Sample preparation: 2 mL Plasma + 800 ng clomipramine in MeOH + 2 mL 1 M NaOH + 5 mL hexane.isoamyl alcohol 99:1, shake mechanically for 15 min, centrifuge at 1686 g for 5 min. Remove the organic phase and add it to 200 |xL 0.05% orthophosphoric acid, shake for 15 min, centrifuge for 5 min, inject a 50 |xL aliquot of the aqueous phase (J.Liq.Chromatogr. 1981, 4, 849).
HPLC VARIABLES

Guard column: 23 X 3.9 Bondapak/Corasil C 18

Column: 300 x 4.6 10 jxm jxBondapak C18 Mobile phase: MeCN:buffer 40:60 (Buffer was 13.68 g KH2PO4 in 2 L water, adjusted to pH 4.7 with dilute KOH.) Column temperature: 50 Flow rate: 2 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Retention time: 4 Internal standard: clomipramine (8.5) Limit of detection: 2 ng

OTHER SUBSTANCES

Extracted: metabolites Simultaneous: amoxapine, amitriptyline, chlordiazepoxide, chlorpromazine, cimetidine, desipramine, diazepam, doxepin, flurazepam, imipramine, lorazepam, oxazepam, pentobarbital, perphenazine, phenobarbital, phenytoin, prochlorperazine, propoxyphene, secobarbital, thioridazine, trifluoperazine Noninterfering: acetaminophen, codeine, meperidine Interfering: nortriptyline
KEYWORDS

plasma
REFERENCE Wong,S.H.Y.; Waugh,S.W. Determination of the antidepressants maprotiline and amoxapine, and their metabolites, in plasma by liquid chromatography, Clin.Chem., 1983, 29, 314-318. SAMPLE

Matrix: blood Sample preparation: Inject 200 |xL serum onto column A and elute with mobile phase A for 10 min then back-flush column A onto column B with mobile phase B for 4 min. Elute column B with mobile phase B and monitor the effluent. Remove column A from circuit and wash with MeCN:water 60:40 for 6 min then with mobile phase A for 10 min.
HPLC VARIABLES

Column: A 40 X 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh) Mobile phase: A 50 mM pH 7.5 potassium phosphate; B MeCN: 100 mM pH 2.7 potassium phosphate 32.5:67.5, containing 0.2 g/L sodium 1-heptanesulfonate Flow rate: 1 Injection volume: 200 Detector: UV 210
CHROMATOGRAM

Retention time: 9 Limit of detection: 10 ng/mL

OTHER SUBSTANCES

Simultaneous: amitriptyline, clomipramine, doxepin, desipramine, imipramine, maprotiline, nortriptyline, trimipramine

KEYWORDS

serum; column-switching; use gradient to determine metabolites

REFERENCE Matsumoto,K.; Kanba,S.; Kubo,H.; Yagi,G.; Iri,H.; Yuki,H. Automated determination of drugs in serum by column-switching high-performance liquid chromatography. IV. Separation of tricyclic and tetracyclic antidepressants and their metabolites, Clin.Chem., 1989, 35, 453-456. SAMPLE

Matrix: blood Sample preparation: 2 mL Plasma + 100 jxL 1 jxg/mL 2-hydroxydesmethylimipramine, mix, add 300 ul. 2 M pH 9.7 carbonate buffer, mix, add 4 mL heptanedsopentyl alcohol 93:7, shake mechanically for 20 min, centrifuge at 1600 g for 5 min. Remove the organic layer and add it to 250 |xL 7 mM orthophosphoric acid, vortex vigorously, centrifuge at 1600 g for 10 min, inject a 100 |xL aliquot of the aqueous layer.
HPLC VARIABLES

Column: 150 X 4.6 5 fjim Spherisorb C6 Mobile phase: 105 |xM nonylamine in MeCN:5 mM KH2PO4 + 14 mM orthophosphoric acid 23:77 Column temperature: 35 Flow rate: 2.2 Injection volume: 100 Detector: UV 210
CHROMATOGRAM

Retention time: 22 Internal standard: 2-hydroxydesmethylimipramine Limit of detection: 2 ng/mL

OTHER SUBSTANCES

Extracted: metabolites, loxapine Simultaneous: propranolol, doxepin, desmethylimipramine, haloperidol, protriptyline, imipramine, amitriptyline Noninterfering: chlorpromazine, clomipramine, maprotiline, nortriptyline, thioridazine, trimipramine, trifluoperazine
KEYWORDS

plasma
REFERENCE Cheung,S.W.; Tang,S.W.; Remington,G. Simultaneous quantitation of loxapine, amoxapine and their 7and 8-hydroxy metabolites in plasma by high-performance liquid chromatography, J.Chromatogr., 1991, 564, 213-221. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 1 mL 0.6 M pH 9.8 carbonate buffer + 40 |ULL 5 |xg/ mL maprotiline in 10 mM HCl + 5 mL 200 g/L ethyl acetate in n-heptane, mix by rocking for 10 min, centrifuge at 1500 g for 10 min. Remove organic layer and add it to 150 |xL 100 mM HCl, mix 10 min, centrifuge at 1500 g for 10 min. Discard organic layer and evaporate aqueous layer at 45 in a vacuum centrifuge for 1 h. Take up residue in 50 |xL 1 M pH 10.3 carbonate buffer and 25 fiL 10 mg/mL dansyl chloride in MeCN, vortex, allow to react at room temperature for 45 min, evaporate at 45 in a vacuum centrifuge for 20 min, reconstitute in 125 |xL MeCN:water 75:25, vortex, centrifuge for 3-5 min, inject a 25-40 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jxm Supelcosil LC-18

Mobile phase: MeCN:25 mM KH2PO4 75:25 + 500 |xL/L orthophosphoric acid + 600 |xL/L n-butylamine Flow rate: 2 Injection volume: 25-40 Detector: F ex 235 em 470 (cut-off)
CHROMATOGRAM

Retention time: 11.18 Internal standard: maprotiline (12.8)

OTHER SUBSTANCES

Simultaneous: fiuoxetine, propranolol, clovoxamine, fluvoxamine, fenfluramine, desipramine, protriptyline, nortriptyline, sertraline, norfluoxetine Noninterfering: amitriptyline, imipramine, clomipramine, trimipramine, mianserin, chlordiazepoxide, trazodone, cyclobenzaprine, nomifensine, bupropion, metoprolol, atenolol, pindolol, tranylcypromine, moclobemide, thioridazine, citalopram, clozapine, carbamazepine, doxepin, loxapine
KEY WORDS

plasma
REFERENCE Suckow,R.E; Zhang,M.R; Cooper,T.B. Sensitive and selective liquid-chromatographic assay of fluoxetine and norfluoxetine in plasma with fluorescence detection after precolumn derivatization, Clin.Chem., 1992, 38, 1756-1761. SAMPLE

Matrix: blood Sample preparation: 2 mL Serum + 75 |JLL MeCN containing 4 mg/mL loxapine + 2 mL 250 mM NaOH + 400 |xL isoamyl alcohol, vortex vigorously, let stand for 5 min, add 10 mL heptane, shake vigorously for 1 h, centrifuge at >2000 g for 30 min. Remove the upper heptane layer and add it to 1 mL 100 mM pH 3 glycylglycine buffer, shake vigorously for 1 h, centrifuge at >2000 g for 30 min. Discard the heptane layer, add 1 mL 250 mM NaOH to the aqueous layer, add 5 mL n-pentane, shake for 1 h, centrifuge at 2000 g for 30 min. Remove the organic layer and evaporate it to dryness under educed pressure, reconstitute the residue in 70 \LL mobile phase, vortex vigorously, centrifuge at 2000 g for 2-3 min, inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 40 jxm pellicular silica (CEL Associates, Houston) Column: 100 X 6 3 |xm silica 80 A (CEL Associates, Houston) Mobile phase: MeCN:buffer 20:80 containing 21 mM n-nonylamine, pH 7.4-7.8 (Buffer was 25 mM Na2HPO4 adjusted to pH 3 with concentrated phosphoric acid.) Flow rate: 1.6 Injection volume: 50 Detector: UV (wavelength not specified)
CHROMATOGRAM

Retention time: 9.63 Internal standard: loxapine (22.68)

OTHER SUBSTANCES

Extracted: doxepin Simultaneous: amitriptyline, chlorpromazine, clomipramine, desipramine, fluoxetine, imipramine, mianserin, nortriptyline, thioridazine, trimipramine Noninterfering: diazepam

KEYWORDS

serum; amoxapine is IS
REFERENCE Adamczyk,M.; Fishpaugh,J.R.; Harrington,C. Quantitative determination of E- and Z-doxepin and Eand Z-desmethyldoxepin by high-performance liquid chromatography, Ther.Drug Monti., 1995, 17, 371-376. SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol:n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 ^m NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 299
CHROMATOGRAM

Retention time: 7.57 Limit of detection: <120 ng/mL

KEYWORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol; aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; tri-

fluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262. SAMPLE

Matrix: blood, tissue Sample preparation: Blood or serum. 1 mL Blood or serum + 1 |xg cianopramine + 1 mL water, vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexane:l-butanol 95:5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it to 100 JJLL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic layer and inject a 30 |xL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver homogenate + 10 |xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexanerl-butanol 95:5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it to 400 |xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic layer and inject a 30 |xL aliquot of the aqueous layer.
HPLC VARIABLES

Guard column: 15 X 3.2 7 jxm RP-18 Newguard (Applied Biosystems) Column: 100 X 4.6 5 |xm Brownlee Spheri-5 RP-18 Mobile phase: MeCN:100 mM NaH2PO4:diethylamine 40:57.5:2.5 Flow rate: 2 Injection volume: 30 Detector: UV 220
CHROMATOGRAM

Retention time: 6.61 Internal standard: cianopramine (8.93)

OTHER SUBSTANCES

Simultaneous: amitriptyline, benztropine, brompheniramine, chlorpheniramine, chlorpromazine, clomipramine, cyproheptadine, desipramine, diphenhydramine, dothiepin, doxepin, haloperidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine, methadone, metoclopramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, northiaden, nortriptyline, pentobarbital, pheniramine, promethazine, propoxyphene, propranolol, protriptyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazodone, trihexyphenidyl, trimipramine, triprolidine Noninterfering: dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine, trifluoperazine Interfering: fluoxetine
KEYWORDS

serum; whole blood; liver

REFERENCE McIntyre,I.M.; King,C.V.; Skafidis,S.; Drummer,O.H. Dual ultraviolet wavelength high-performance liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and some selected metabolites, J.Chromatogr., 1993, 621, 215-223. SAMPLE

Matrix: blood, tissue, urine Sample preparation: Serum, urine. 500 |xL Serum or urine + 100 |xL 2 |xg/mL diazepam + 200 |xL 20% sodium carbonate + 500 |xL water + 3 mL n-hexane:isoamyl alcohol 98.5: 1.5, mix for 2 min, centrifuge at 1200 g for 5 min. Remove the organic phase and evaporate it under a gentle stream of nitrogen at about 40. Dissolve the residue in 100 JJLL mobile phase, inject a 10 |xL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 |xg/mL diazepam, centrifuge at 15 000 g for 10 min. Add 500 |xL 20% sodium carbonate and 4 mL n-hexane:isoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic phase and evaporate it under a gentle stream of nitrogen at about 40. Dissolve the residue in 100 |xL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 2 fxm TSK gel Super-Octyl (A) or 100 X 4.6 5 |xm Hypersil MOS-C8 (B), (Yokogawa, Japan) Mobile phase: MeOH:20 mM pH 7 KH2PO4 60:40 Flow rate: 0.6 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 5.6 (A), 7.8 (B) Internal standard: diazepam (4.4, A) Limit of quantitation: 50 ng/mL (serum, urine) (A), 500 ng/mL (tissue) (A)
OTHER SUBSTANCES

Extracted: amitriptyline, clomipramine, dothiepin, doxepin, imipramine, melitracen, mianserin, nortriptyline Noninterfering: barbital, carbamazepine, ethosuximide, hexobarbital, lofepramine, pentobarbital, phenobarbital, phenytoin, primidone, sulpiride, trimethadione, trimipramine Interfering: desipramine, maprotiline
KEYWORDS

serum; brain; liver

REFERENCE Tanaka,E.; Terada,M.; Nakamura,T.; Misawa,S.; Wakasugi,C. Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 |xm porous microspherical silica gel, J.Chromatogr.B, 1997, 692, 405-412. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JJLL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)

HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 211.1 CHROMATOGRAM Retention time: 14.187
KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performahce liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763, 149-163. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Econosil C8 Mobile phase: MeCN:buffer 30:70 (Buffer was 20 mM KH2PO4 and 14 mM triethylamine adjusted to pH 3.0 with phosphoric acid.) Injection volume: 20 Detector: UV 210
CHROMATOGRAM

Retention time: 5.5 Limit of quantitation: < 1000 ng/mL

OTHER SUBSTANCES

Simultaneous: amitriptyline, carbamazepine, imipramine, nortriptyline Also analyzed: doxepin, desipramine, protriptyline, cyclobenzaprine, maprotiline
KEYWORDS

UV spectra given
REFERENCE Ryan,T.W. Identification and quantification of tricyclic antidepressants by UV-photodiode array detection with multicomponent analysis, J.Liq.Chromatogr., 1993, 16, 1545-1560. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min.

Column temperature: 30 Flow rate: 2 Detector: UV 210

OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pjndthyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994, 18, 233-242. SAMPLE

Matrix: solutions

HPLC VARIABLES

Column: 250 X 4.6 5 fim Supelcosil LC-DP (A) or 250 X 4 5 jxm LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acid.buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 11.40 (A), 5.55 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119.

Amoxicillin
Molecular formula: C16H19N3O5S Molecular weight: 365.41 CAS Registry No.: 26787-78-0 (anhydrous), 61336-70-7 (trihydrate) Lednicer No.: 4 179, 180 SAMPLE

Matrix: blood Sample preparation: Vortex plasma sample for 15 s, centrifuge at 4000 rpm for 10 min at 4 . 250 |xL Plasma + 250 jxL 5 |xg/mL cefadroxil in 100 mM pH 2.5 KH2PO4, centrifuge at 4800 g for 30 min. Inject a 150 |xL aliquot of the clear supernatant onto column A and elute to waste with mobile phase B, after 15 min elute the contents of column A onto column B with mobile phase A, after 5 min remove column A from the circuit, elute column B with mobile phase A, monitor effluent from column B. Re-equilibrate column A with mobile phase B.
HPLC VARIABLES

Column: A 40 X 4.6 10 |xm Nucleosil 100 C18; B 250 X 4.6 5 |xm Spherisorb ODS II Mobile phase: A MeOH: 10 mM sodium heptanesulfonate and 30 mM NaH2PO4 25:75 adjusted to pH 2.5 with phosphoric acid; B MeOH: 10 mM sodium heptanesulfonate and 30 mM NaH2PO4 8:92 adjusted to pH 2.5 with phosphoric acid Flow rate: 1.5 Injection volume: 150 Detector: UV 230
CHROMATOGRAM

Retention time: 34.8-38.8 Internal standard: cefadroxil (31.8-32.8) Limit of quantitation: 50.1 ng/mL
KEY WORDS

plasma; column-switching; pharmacokinetics

REFERENCE Muth,R; Metz,R.; Beck,H.; Bolten,W.W.; Vergin,H. Improved high-performance liquid chromatographic determination of amoxicillin in human plasma by means of column switching, J.Chromatogr.A, 1996, 729, 259-266. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |ULL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters)

Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 3.067 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A,

1997, 763, 149-163.

SAMPLE

Matrix: milk Sample preparation: Mix 10 mL milk with 2 mL 100 mM tetraethylammonium chloride, add 40 mL MeCN slowly with continual stirring, let stand for 10 min, decant the supernatant through a plug of glass wool. Collect 40 mL filtrate, add 2 mL buffer, evaporate to 1-2 mL under reduced pressure at 40-50, dilute to 4 mL with water, filter (0.45 |xm PVDF). Inject a 2 mL aliquot onto a 150 X 4.6 5 jxm Supelcosil LC-18 column, elute with MeCN:10 mM KH2PO4 0:100 for 3 min, to 60:40 over 37 min at 1 mL/min, collect a 1.52 mL aliquot containing the compound (ca. 12.5 min), evaporate to <1 mL under reduced pressure, add 200 (xL 10 mM KH2PO4 containing 10 mM phosphoric acid and 10 mM sodium decanesulfonate, make up to 1 mL with water, inject an aliquot. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM Na2HPO4 in a 5:1 ratio, pH 6.)
HPLC VARIABLES

Column: 150 X 4.6 5 jxm Supelcosil LC-18 Mobile phase: MeCN:buffer 32:68 (Buffer was 15 mM phosphoric acid containing 7.5 mM sodium dodecyl sulfate.) Flow rate: 1 Injection volume: 200 Detector: UV 215
REFERENCE Moats,W.A.; Romanowski,R.D. Multiresidue determination of (3-lactam antibiotics in milk and tissues with the aid of high-performance liquid chromatographic fractionation for clean up, J.Chromatogr.A, 1998, 812, 237-247. SAMPLE

Matrix: milk Sample preparation: Condition a 500 mg tC18 SPE cartridge (Waters) with 20 mL MeOH, 20 mL water, and 10 mL 2% NaCl. Centrifuge 30 mL milk at 1500 g for 10 min. Dilute a 10 mL portion of the defatted milk with 20 mL water, add 200 JJIL 2 u-g/mL penicillin V in pH 9.0 buffer, add 6 mL 170 mM sulfuric acid, add 5.6 mL 5% sodium tungstate, shake vigorously for 1 min, allow to stand for 5 min, check that the pH is in the range 4.6-4.8 (if it is outside this range start again using a different volume of sodium tungstate solution), centrifuge at 1500 g for 10 min, adjust the pH of the supernatant to 8.1-8.2 with 5 M and 0.1 M NaOH, filter (glass fiber) the clear liquid phase. Pass the filtrate through the SPE cartridge at 2 mL/min, wash with 2 mL water, dry by pulling air through

the cartridge for 1 min, elute with 2 mL MeCN. Add 150 |xL pH 9.0 buffer to the eluate and evaporate to about 100 |xL under a stream of nitrogen at 45-50, add 400 |xL pH 9.0 buffer, add 75 JJLL reagent I, vortex for 30 s, let stand at room temperature for 10 min, use 500 |xL water to transfer the mixture to a separatory funnel, add 20 mL dichloromethane, add 5 mL pH 2.45 buffer, shake for 1 min, let stand for no more than 5 min. Remove the organic layer and evaporate it to dryness under reduced pressure at 35-40, dissolve the residue in 500 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex for 30 s, let stand at room temperature for 10 min, add 450 JJLL reagent II, vortex for 1 min, heat at 55 1 for 30 min, cool, filter (0.45 jjum), inject a 150 |xL aliquot. (Prepare pH 9.0 buffer by dissolving 0.34 g KH2PO4 in water, adjusting the pH to 9.0 with NaOH, and making up to 100 mL with water. Prepare pH 2.45 buffer by dissolving 2.72 g KH2PO4 in water, adjusting the pH to 2.45 with phosphoric acid, and making up to 100 mL with water. Prepare reagent 1 by dissolving 1.13 g benzoic anhydride in MeCN, make up to 25 mL with MeCN. Prepare reagent II by dissolving 6.905 g 1,2,4-triazole in 30 mL water and adding 5 mL 26 mM mercuric chloride in water, adjust pH to 9.0 0.05 with 5 M NaOH, make up to 50 mL. Prepare reagents I and II 1-4 h before use. Silanize glassware with dichlorodimethylsilane.)
HPLC VARIABLES

Column: 150 x 3.9 4 fim Nova-Pak C18 Mobile phase: Gradient. A as MeCN:buffer 10:90. B was MeCNrbuffer 30:70. A:B from 100: 0 to 0:100 over 30 min, maintain at 0:100 for 13 min, return to initial conditions over 2 min, re-equilibrate at initial conditions for 5 min. (Prepare buffer by dissolving 9.938 g Na2HPO4, 17.938 g NaH2PO4.H2O, and 4.964 g sodium thiosulfate in water, make up to 2 L with water, pH 6.5.) Column temperature: 30 Flow rate: 1 Injection volume: 150 Detector: UV 323
CHROMATOGRAM

Retention time: 25 Internal standard: penicillin V (28.5) Limit of detection: 1.4 ng/mL Limit of quantitation: 2.0 ng/mL
OTHER SUBSTANCES

Extracted: ampicillin, cloxacillin, dicloxacillin, oxacillin, penicillin G

KEYWORDS

derivatization; cow; SPE

REFERENCE Sorensen,L.K.; Rasmussen,B.M.; Boison,J.O.; Keng,L. Simultaneous determination of six penicillins in cows' raw milk by a multiresidue high-performance liquid chromatographic method, J.Chromatogr.B, 1997, 694, 383-391. SAMPLE

Matrix: perfusate Sample preparation: Vortex perfusate, centrifuge at 11600 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES

Guard column: 20 X 2 5 |xm Hypersil ODS Column: 150 X 4.6 5 fim Hypersil ODS Mobile phase: MeOH:buffer 10:90 (Buffer was 50 mM KH2PO4 containing 0.1% triethylamine adjusted to pH 3 with orthophosphoric acid.) Flow rate: 1

Injection volume: 100 Detector: UV 230

CHROMATOGRAM

Retention time: 6.7 Limit of detection: 20 ng/mL Limit of quantitation: 100 ng/mL
REFERENCE Erah,P.O.; Barrett,D.A.; Shaw,P.N. Reversed-phase high-performance liquid chromatographic assay methods for the analysis of a range of penicillins in in vitro permeation studies, J.Chromatogr.B, 1998, 705, 63-69. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere C18 Mobile phase: MeCN: 10 mM pH 6.1 potassium phosphate buffer 5:95 Flow rate: 1 Detector: UV 201
REFERENCE Walter,E.; Janich,S.; Roessler,B.J.; Hilfinger,J.M.; Amidon,G.L. HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: In vitro-in vivo correlation with permeability data from rats and humans, J.Pharm.Sci., 1996, 85, 1070-1076. SAMPLE

Matrix: solutions Sample preparation: Prepare amoxicillin sodium solutions in water adjusted to pH 4 with trifluoroacetic acid.
HPLC VARIABLES

Column: 150 X 4.6 5 juim Supelcosil ABZ+plus (Supelco) Mobile phase: Gradient. A MeCN:0.1% pH 2.1 trifluoroacetic acid 7:93; B MeCN:0.1% pH 2.1 trifluoroacetic acid 20:80. A:B 100:0 for 13 min, from 100:0 to 40:60 in 2 min, maintain at 40:60 for 30 min, from 40:60 to 100:0 in 2 min, maintain at 100:0 for 13 min Flow rate: 1 Injection volume: 5 Detector: UV 230; MS, PE-Sciex API I, ionspray interface at 5500 V, orifice potential voltage 50 V, m/z 100-800 CHROMATOGRAM Retention time: 9.99 OTHER SUBSTANCES Simultaneous: impurities
REFERENCE Valvo,L.; Ciranni,E.; Alimenti,R.; Alimonti,S.; Draisci,R-5 Giannetti,L.; Lucentini,L. Development of a simple liquid chromatographic method with UV and mass spectrometric detection for the separation of substances related to amoxicillin sodium, J.Chromatogr.A, 1998, 797, 311-316. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 |xL aliquot of a 100-500 jxg/mL solution in mobile phase.

HPLC VARIABLES

Column: 100 X 4.6 5 |xm Hypersil C8 MOS 10OA coated with phosphatidylcholine (95% pure soybean lecithin, Epikuron, Lucas Meyer & Co.) (Coat column by recycling a 1 mM solution of phosphatidylcholine in MeOHrwater 80:20 for 24 h.) Mobile phase: MeCN:35 mM pH 7.4 sodium phosphate buffer 40:60 Flow rate: 0.5-2 Injection volume: 20 Detector: UV 254
CHROMATOGRAM Retention time: k' 0.85 OTHERSUBSTANCES

Also analyzed: antipyrine, carbamazepine, chlorpheniramine, chlorpromazine, clonidine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, flufenamic acid, haloperidol, hydroxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone, nadolol, phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole, testosterone, thioridazine, tolfenamic acid, verapamil Noninterfering: acetaminophen, aspirin, azathioprine, caffeine, carprofen, chlorambucil, cimetidine, fenoterol, flurbiprofen, ibuprofen, ketoprofen, ranitidine, salicylic acid, sulfamethoxazole, theophylline, thioguanine, tiaprofenic acid, trimethoprim, valproic acid
KEYWORDS

comparison with capillary electrophoresis

REFERENCE Hanna,M.; de Biasi,V.; Bond,B.; Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics of drugs: A comparison of a large and diverse drug series utilizing chromatographic and electrophoretic methodology, Anal. Chem., 1998, 70, 2092-2099. SAMPLE

Matrix: tissue Sample preparation: Condition a 500 mg Isolute SCX SPE cartridge (Jones Chromatography, Hengoed, UK) with MeOH and water. Condition a 100 mg PGC (porous graphitic carbon) SPE cartridge (Shandon, Runcorn, UK) with acetone and pH 7.7 borate buffer. Add 20 mL water to 5 g tissue, homogenize, add 5 mL 170 mM sulfuric acid and 5 mL 5% aqueous sodium tungstate, mix well, centrifuge at 14000 g for 5 min. Discard pellet, add six drops orthophosphoric acid to the supernatant to adjust the pH to 2-2.5. Add it to the SCX SPE cartridge, allow to flow through the cartridge under vacuum at 2 mL/ min, wash with 5 mL 10 mM sulfuric acid, elute with 10 mL pH 7.7 borate buffer. Add the eluate to the PGC SPE cartridge. Wash with 5 mL water, place in-line filter (0.2 |xm, Anotop) below cartridge, elute with 20 mL acetone. Evaporate to dryness. Add 500 JULL water to the dry residue, add 20 |xL 2% acetic anhydride in MeCN and let stand for 3 min. Add 500 |xL triazole/mercuric chloride derivatizing reagent and heat the mixture at 65 for 20 min. Inject a 100 JJLL aliquot. (Borate buffer was 200 mM boric acid adjusted to pH 7.7 with 40% NaOH solution. Triazole/mercuric chloride reagent was prepared by mixing 34.45 g 1,2,4-triazole with 150 mL water and 25 mL 10 mM mercuric chloride, adjusted to pH 9.0 with 1 M NaOH and made up to 250 mL with water.) GL - 5 (xm Kromasil KR 100 C8 (Hichrom)
HPLC VARIABLES

Column: 250 X 3.2 5 |xm Kromasil KR 100 C8 (Hichrom) Mobile phase: MeCN:buffer 20:80 (Buffer was 15 mM potassium dihydrogen phosphate and 15 mM sodium thiosulfate) Flow rate: 0.55 Injection volume: 100 Detector: UV 325

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CHROMATOGRAM

Retention time: 10.5 Limit of detection: 5 jig/kg (muscle) Limit of quantitation: 50 fxg/kg (muscle), 100 |xg/kg (liver)
OTHER SUBSTANCES

Extracted: ampicillin
KEYWORDS

SPE; cow; liver; muscle; derivatization

REFERENCE Rose,M.D.; Tarbin,J.; Farrington,W.H.; Shearer,G. Determination of penicillins in animal tissues at trace residue concentrations: II. Determination of amoxicillin and ampicillin in liver and muscle using cation exchange and porous graphitic carbon solid phase extraction and high-performance liquid chromatography, Food Addit.Contam., 1997, 14, 127-133. SAMPLE

Matrix: tissue Sample preparation: Condition a 3 mL 500 mg Sep-Pak C18 SPE cartridge with 5 mL MeOH, 2 mL water, and 2 mL 2% trichloroacetic acid. Homogenize (Ultra-Turrax T25) 5 g blended tissue with 20 mL 10 mM pH 4.5 phosphate buffer at 10000 rpm for 1.5 min, centrifuge at 4500 rpm for 10 min, decant supernatant, homogenize residue with another 20 mL buffer, centrifuge. Combine the supernatants and filter them through glass wool, add 1 mL 75% trichloroacetic acid to the filtrate, vortex for 30 s, centrifuge at 4500 rpm for 20 min, filter the supernatant through glass wool. Add the filtrate to the SPE cartridge at 1-2 mL/min, wash with 2 mL 2% trichloroacetic acid, wash with 2 mL water, elute with 1.5 mL MeCN at 0.7 mL/min. Add the eluate to 500 |xL water and 3 mL ethyl ether, vortex gently for 30 s, centrifuge at 2000 rpm for 3 min, discard the organic layer. Add 200 |xL 20% trichloroacetic acid solution to the aqueous phase, vortex for 15 s, add 200 |xL 7% formaldehyde in 400 mM citric acid, vortex for 30 s, heat in boiling water bath for 30 min, cool to room temperature, add 500 mg NaCl, mix briefly, add 3 mL ethyl ether, vortex for 1 min, centrifuge at 2000 rpm for 3 min, repeat extraction twice more. Combine the organic layers and evaporate them to dryness under a stream of nitrogen at 40, reconstitute the residue in 500 JJLL mobile phase, vortex thoroughly, inject a 50 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 S5 ODS2 Mobile phase: MeCN.buffer 20:80 (Buffer was 50 mM KH2PO4 adjusted to pH 5.6 with KOH.) Flow rate: 1 for 10 min then 2 Injection volume: 50 Detector: F ex 358 em 440 CHROMATOGRAM Retention time: 6 Limit of detection: 0.5 ppb (catfish), 0.8 ppb (salmon) Limit of quantitation: 1.2 ppb (catfish), 2.0 ppb (salmon)
KEYWORDS

derivatization; fish; catfish; salmon; SPE

REFERENCE Ang,C.Y.W.; Luo,W.; Hansen,E.B.,Jr.; Freeman,J.P.; Thompson,H.C.,Jr. Determination of amoxicillin in catfish and salmon tissues by liquid chromatography with precolumn formaldehyde derivatization, JAOACInL, 1996, 79, 389-396.

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Amphetamine
Molecular formula: C9H13N Molecular weight: 135.21 CAS Registry No.: 300-62-9, 139-10-6 (phosphate), 60-13-9 (sulfate), 1407-85-8 (d-form tannate) Merck Index: 623 Lednicer No,: 1 37, 70; 2 47

SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 1 mL 100 mM NaOH + 3 mL n-hexane, shake for 20 min, centrifuge for 10 min. Remove 2 mL of the organic layer and evaporate it to dryness using a vacuum centrifuge, reconstitute the residue in 500 |xL 100 |xg/mL (S)-(+)benoxaprofen chloride in dried dichloromethane, let stand at room temperature for 30 min, inject a 10 jxL aliquot. (Synthesis of benoxaprofen chloride is as follows. Dissolve 600 mg benoxaprofen in 50 mL toluene, slowly add 5 mL freshly-distilled thionyl chloride, reflux for 30 min, evaporate to dryness, recrystallize benoxaprofen chloride from dichloromethane.)
HPLC VARIABLES

Column: 250 X 4.6 7 |xm Zorbax-Sil Mobile phase: Cyclohexane:dichloromethane:THF 50:10:10 Flow rate: 1 Injection volume: 10 Detector: F ex 312 em 365
CHROMATOGRAM

Retention time: 8.0 (R-(-)), 9.5 (S-(+))

OTHER SUBSTANCES

Extracted: methamphetamine, tranylcypromine

KEY WORDS

plasma; derivatization; normal phase; chiral

REFERENCE Weber,H.; Spahn,H.; Mutschler,E.; Mohrke,W. Activated a-alkyl-a-arylacetic acid enantiomers for stereoselective thin-layer chromatographic and high-performance liquid chromatographic determination of chiral amines, J.Chromatogr., 1984, 307, 145-153. SAMPLE

Matrix: blood Sample preparation: 100 |xL Serum + 50 JJIL 100 ng/mL aniline sulfate in water + 200 |xL 20 mM pH 10.6 carbonate buffer + 2 mL ethyl acetate, shake for 15 min, centrifuge at 1200 g for 5 min. Remove the organic layer and add it to 200 (JLL 50 mM HCl, shake for 15 min, centrifuge at 1200 g for 5 min. Remove the aqueous layer and add it to 40 \LL 250 mM NaOH, add 50 JJLL 330 mM pH 7.8 phosphate buffer, add 250 ^L MeCN, add 25 (JLL 1 mM (-)-l-(9-fluorenyl)ethyl chloroformate in acetone, let stand overnight at room temperature, add 30 JULL 100 mM glycine in water, add 750 |xL n-pentane, vortex for 2 min, centrifuge at 1200 g for 5 min. Remove the organic layer and evaporate it to dryness under vacuum, reconstitute the residue in MeCN:water 50:50, inject a 100 JJLL aliquot.
HPLC VARIABLES

Guard column: Direct-Connect column prefilter (Alltech) Column: 150 X 4.6 3 |xm Adsorbosphere HS C18 (Alltech)

Mobile phase: MeCN:THF:20 mM pH 3.6 acetate buffer 39:15:46 Flow rate: 1 Injection volume: 100 Detector: F ex 265 em 330 CHROMATOGRAM Retention time: 22.6 (R), 23.6 (S) Internal standard: aniline (21.0) Limit of quantitation: 5 ng/mL OTHER SUBSTANCES Extracted: methamphetamine
KEYWORDS

serum; rat; chiral; derivatization

REFERENCE Hutchaleelaha,A.; Walters,A-; Chow,H.-H.; Mayersohn,M. Sensitive enantiomer-specific high-performance liquid chromatographic analysis of methamphetamine and amphetamine from serum using precolumn fluorescent derivatization, J.ChromatognB, 1994, 658, 103-112. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 1 mL 500 mM pH 11 borate buffer, mix, add 2.5 mL diethyl ether, vortex for 5 min, centrifuge at 1200 g for 5 min, remove organic layer, repeat extraction. Combine the organic layers and add them to 200 |xL 100 mM HCl, vortex for 2 min, centrifuge at 1200 g for 5 min. Remove the aqueous phase and add it to 150 (xL 1 M pH 8 borate buffer and 100 uL 4 mM 9-fluorenylmethyl chloroformate in MeCN, shake, allow to react at 50 for 5 min, add 20 jxL 20 mM proline in water, allow to react at 50 for 2 min, inject a 200 (xL aliquot.
HPLC VARIABLES

Column: 150 X 3.9 4 am Nova-Pak phenyl Mobile phase: MeCN:50 mM pH 6.0 sodium phosphate buffer 50:50 Flow rate: 1 Injection volume: 200 Detector: F ex 260 em 315
CHROMATOGRAM

Retention time: 12 Limit of quantitation: 0.5 ng/mL

OTHER SUBSTANCES

Extracted: methamphetamine, desmethyldeprenyl

KEYWORDS

plasma
REFERENCE La Croix,R.; Pianezzola,E.; Strolin Benedetti,M. Sensitive high-performance liquid chromatographic method for the determination of the three main metabolites of selegiline (L-deprenyl) in human plasma, J.Chromatogr.B, 1994, 656, 251-258. SAMPLE

Matrix: blood, tissue, dialysate Sample preparation: Plasma. 45 jxL Plasma + 5 |xL 40 |xM tryptamine + 100 |xL 100 mM borate buffer adjusted to pH 10.6 with NaOH + 200 |xL ethyl acetate, vortex for 2 min,

let sit on ice for 10 min, add 200 |JLL ethyl acetate, add 200 jxL water, vortex briefly, centrifuge at 4 at 18000 g for 10 min. Remove 200 jxL of the organic supernatant and evaporate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 80 fiL 50 mM KH2PO4 adjusted to pH 2.6 with phosphoric acid, add 20 |xL borate buffer adjusted to pH 11.5 with NaOH, add 20 JJLL reagent, let stand for at least 2 min, keep at 4, inject a 75 |xL aliquot. Tissue. Sonicate brain tissue with 9 volumes of 8 |xM tryptamine in 100 mM pH 10.6 borate buffer for 20 s, centrifuge at 4 at 18000 g for 10 min, remove a 100 JLJLL aliquot of the supernatant and add 200 |xL ethyl acetate, vortex for 2 min, let sit on ice for 10 min, add 200 |xL ethyl acetate, add 200 jxL water, vortex briefly, centrifuge at 4 at 18000 g for 10 min. Remove 200 jxL of the organic supernatant and evaporate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 80 (xL 50 mM KH2PO4 adjusted to pH 2.6 with phosphoric acid, add 20 |xL borate buffer adjusted to pH 11.5 with NaOH, add 20 fiL reagent, let stand for at least 2 min, keep at 4, inject a 75 \JJJ aliquot. Dialysate. 100 |xL Dialysate + 20 |xL reagent, let stand for at least 2 min, keep at 4, inject a 75 JLJLL aliquot. (Reagent was 27 mg o-phthaldialdehyde in 500 |JLL EtOH, add 5 mL 100 mM pH 9.6 borate buffer, add 40 |xL 3-mercaptopropionic acid, refrigerate, use for up to 4 days.)
HPLC VARIABLES

Guard column: 20 X 2 37-50 fim Bondapak C18/Corasil Column: 250 X 4.6 5 |xm LC-18 (Supelco) Mobile phase: Gradient. MeOH:buffer 35:65 for 3 min, to 65:35 over 1 min, maintain at 65:35 for 14 min, return to initial conditions over 1 min, re-equilibrate for 6 min. (Buffer was 50 mM KH2PO4 adjusted to pH 5.5 with KOH.) Flow rate: 1.5 Injection volume: 75 Detector: F ex 340 em 440
CHROMATOGRAM

Retention time: 14.5 Internal standard: tryptamine (13) Limit of detection: 200 pg (plasma, tissue), 50 pg (dialysate) Limit of quantitation: 100 pg (dialysate)
OTHER SUBSTANCES

Extracted: metabolites, p-hydroxyamphetamine

KEYWORDS

rat; brain; plasma; derivatization

REFERENCE Bowyer,J.E; Clausing,R; Newport,G.D. Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid, J.Chromatogr.B, 1995, 666, 241-250. SAMPLE

Matrix: blood, urine Sample preparation: Adjust pH of 10 mL plasma or 20 mL urine to 11.4 with 5 M NaOH, add to a column containing 1.5 g Amberlite XAD-2, wash with 10 mL water, elute with 20 (plasma) or 40 (urine) mL chloroformrisopropanol 75:25, add 100 |xL 6 M HCl in EtOH to the eluate, evaporate to dryness under reduced pressure, reconstitute with 1 mL 8% sodium bicarbonate, add 1 mL 0.5% sodium l,2-naphthoquinone-4-sulfonate, heat at 70 for 20 min, add an equal volume of chloroform, vortex for 1 min, inject a 50 fiL aliquot of the organic layer.
HPLCVARIABLES

Column: 150 X 5 Partisil 5 Mobile phase: Hexanexhloroformrethyl acetate:EtOH 50:25:35:1

Column temperature: 20 Flow rate: 2.5 Injection volume: 50 Detector: UV 248

CHROMATOGRAM

Retention time: 5 Internal standard: phenylethylamine (6) Limit of detection: 2 ng

OTHER SUBSTANCES

Extracted: hydroxyamphetamine, methamphetamine, norephedrine

KEYWORDS

derivatization; plasma; normal phase; comparison with other derivatization reagents and with ion-pair chromatography; SPE
REFERENCE Farrell,B.M.; Jefferies,T.M. An investigation of high-performance liquid chromatographic methods for the analysis of amphetamines, J.Chromatogr., 1983, 272, 111-128. SAMPLE

Matrix: blood, urine Sample preparation: Adjust pH of 10 mL plasma or 20 mL urine to 11.4 with 5 M NaOH, add to a column containing 1.5 g Amberlite XAD-2, wash with 10 mL water, elute with 20 (plasma) or 40 (urine) mL chloroformdsopropanol 75:25, add 100 |xL 6 M HCl in EtOH to the eluate, evaporate to dryness under reduced pressure, reconstitute with 1 mL EtOH, add 1 mL reagent, mix, filter, inject a 50 |xL aliquot. (Prepare reagent by dissolving 200 mg o-phthalaldehyde in 2 mL MeOH, add 400 \iL mercaptoethanol, add to buffer, store in the dark in the refrigerator, discard after 5 days. Prepare buffer by dissolving 1 g boric acid in 38 mL water and adjusting pH to 10.4 with 4 M KOH.)
HPLC VARIABLES

Column: 200 X 5 10 |xm Partisil ODS-2 Mobile phase: MeOH:water 73:27 containing 0.2% EDTA Column temperature: 20 Flow rate: 1.8 Injection volume: 50 Detector: F ex 345 em 445
CHROMATOGRAM

Retention time: 9 Internal standard: benzylamine (6) Limit of quantitation: 500 ng/mL
OTHER SUBSTANCES

Extracted: hydroxyamphetamine, norephedrine

KEYWORDS

derivatization; plasma; comparison with other derivatization reagents and with ion-pair chromatography; SPE
REFERENCE Farrell,B.M.; Jefferies,T.M. An investigation of high-performance liquid chromatographic methods for the analysis of amphetamines, J.Chromatogr., 1983, 272, 111-128. SAMPLE

Matrix: blood, urine

Sample preparation: Serum. 1 mL Serum + 1 mL 120 mM sodium dodecyl sulfate in 100 mM NaOH, homogenize, filter (45 fim). Inject on to column A at 180 jxL/min 200 JULL 60 mM sodium dodecyl sulfate in MeCN:water 50:50, 25 |xL filtrate, and 25 |JLL 60 mM sodium dodecyl sulfate in MeCN:water 10:90, wait for 1 min, inject 100 |xL 60 mM sodium dodecyl sulfate in MeCN:water 10:90, inject 200 JJLL 10 mM sodium dodecyl sulfate in MeCN:water 10:90, backflush the contents of column A on to column B with mobile phase, after 18 s remove column A from the circuit, elute column B with mobile phase, monitor the effluent from column B. Wash column A with 300 JJIL 60 mM sodium dodecyl sulfate in MeCN:water 50:50 and 300 fiL 10 mM sodium dodecyl sulfate in MeCN:water 80:20. Urine. 5 mL Urine + 4.5 mL MeCN + 500 |xL 1 M KOH, centrifuge at 2500 rpm for 10 min, filter (45 |xm) the supernatant. Inject on to column A at 180 |xL/min 25 |xL 50 mM KOH in MeCN:water 20:80, 50 JJLL filtrate, 25 |xL 50 mM KOH in MeCN:water 20:80, and 200 fiL MeCN: water 20:80, backflush the contents of column A on to column B with mobile phase, after 18 s remove column A from the circuit, elute column B with mobile phase, monitor the effluent from column B. Wash column A with 400 JJIL MeCN.
HPLC VARIABLES

Column: A 20 X 2 polymeric reagent; B 250 X 4.6 5 |xm Supelcosil LC-18-DB (with a guard column) (Prepare polymeric reagent as follows. Prepare a porous rigid resin using a divinylbenzene:ethylstyrene:styrene 24:6:70 mixture with trimethylsilyl modified silica (102 A average pore size, 1.08 mL/g pore volume, 366 m2/g surface area, 16-20 |xm irregular particle shape, IMPAQ RG 1020 Si silica, PQ Co., Valley Forge PA). Further preparation details are not given but a typical procedure given in the cited reference is as follows. Aerate a mixture of 10 g modified silica in 100 mL water with nitrogen for 15 min, add 10 mL styrene:80% divinylbenzene:t-butyl peroxybenzoate 49:49:2 (remove preservative by passing through a butylcatechol remover (Scientific Polymer, Ontario NY), shake vigorously at room temperature for 4 h, add 150 mL 0.75% polyvinyl alcohol, shake for 4 h, heat at 120 for 24 h while shaking on a Parr instrument, cool to room temperature, filter, wash with 100 mL water, wash with 50 mL MeOH. Add the solid to 500 mL 3 M NaOH in MeOH:water 40:60, shake at room temperature for 14 h (to dissolve the silica), filter, wash with water until the washings are neutral, wash with 100 mL MeOH, dry at 60. The polymer has similar properties to the template silica (US Pat. 4 933 372 (1990)). Soxhlet extract the resin with dioxane for 8 h (Caution! Dioxane is a carcinogen!). Add 25 g aluminum trichloride in 300 mL dry nitrobenzene to 50 g resin and 100 g 4-chloro3-nitrobenzoyl chloride, stir mechanically at 60 for 5 h, pour into a mixture of 150 mL DMF, 100 mL concentrated HCl, and 150 g ice, filter. Wash the solid with 300 mL portions of DMF:water 75:25 until the washings are colorless, wash with warm (60) DMF, wash with six 300 mL portions of dichloromethane:MeOH 2:1. Stir the product in 130 mL 40% benzyltrimethylammonium hydroxide in water, 130 mL water, and 260 mL dioxane at 90 for 8 h, filter, repeat the process. Wash the product with four portions of warm (60) dioxane. Stir the solid with 30 mL acetic acid for 15 min, filter. Wash the solid with dioxane until the washings are neutral, wash with six 300 mL portions of dichloromethane.MeOH 2:1 to give a nitrobenzophenol-substituted polymer (J. Org. Chem. 1984, 49, 924). Heat 4 g 9-fluoreneacetic acid, 3.9 mL oxalyl chloride, 30 mL benzene (dried over anhydrous sodium sulfate, Caution! Benzene is a carcinogen!), and 3 drops of triethylamine at 55 for 1 h, evaporate under reduced pressure to remove oxalyl chloride, dissolve the product in 35 mL dichloromethane to give a 120 mg/mL solution of 9-fluoreneacetyl chloride, dilute to obtain a 2 mM solution. Stir 1.3 g nitrobenzophenol-substituted polymer, 4.2 mL 2 mM 9-fluoreneacetyl chloride solution, 300 |xL triethylamine, and 20 mL dichloromethane at room temperature for 1 h, filter, wash with three 20 mL portions of MeCN to obtain the reagent, polymer-bound nitrobenzophenol 9-fluoreneacetate (J. Chromatogr. 1992, 609, 103).) Mobile phase: Gradient. MeCN:water 50:50 for 3.5 min, to 70:30 over 12 min, maintain at 70:30 for 2.5 min, return to initial conditions over 1 min. (Place a 100 X 4.6 30-40 |xm silica column before the injector.) Column temperature: 60 (column A only) Injection volume: 25-50 Detector: F ex 254 em 305-395

CHROMATOGRAM

Retention time: 9
Limit of quantitation: 25 ng/mL (urine), 600 ng/mL (serum)
OTHER SUBSTANCES

Extracted: methamphetamine (only in urine)

KEYWORDS

derivatization; serum
REFERENCE Bourque,A.J.; KrullJ.S.; Feibush,B- Automated HPLC analyses of drugs of abuse via direct injection of biological fluids followed by simultaneous solid-phase extraction and derivatization with fluorescence detection, Biomed.Chromatogr., 1994, 8, 53-62. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLCVARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 \im Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 3.71 KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: bulk Sample preparation: Mix a 1 mg/mL solution in 1 M sodium carbonate with 2 mL 5 mg/ mL 8-quinolinesulfonyl chloride in acetone, heat at 65 for 20 min, cool, extract twice with 30 mL portions of chloroform. Combine the extracts and dry them over anhydrous magnesium sulfate, evaporate to dryness under a stream of air, reconstitute, inject an aliquot.

HPLC VARIABLES

Guard column: 70 X 2.1 CorPell ODS Column: 300 X 3.9 jxBondapak C18 Mobile phase: MeCN:water:acetic acid 40:59:1 Flow rate: 1.5 Detector: UV 254, UV 280 CHROMATOGRAM Retention time: 19
OTHER SUBSTANCES

Simultaneous: ephedrine, methamphetamine, phenmetrazine, phentermine, phenylpropanolamine, pseudoephedrine

KEY WORDS

derivatization
REFERENCE Noggle,F.T.,Jr.; Clark,C.R. Liquid chromatographic determination of primary and secondary amines as 8-quinolinesulfonyl chloride derivatives, JAssoc.Off.Anal.Chem., 1984, 67, 687-691. SAMPLE

Matrix: bulk Sample preparation: Mix 200 fjimole amine with 500 ixmole (lS)-(+)-camphor-10-sulfonyl chloride, 10 mL diethyl ether, and 10 mL 1 M NaOH, stir vigorously for 1 h, acidify with concentrated HCl, extract three times with diethyl ether. Combine the organic layers and wash them three times with water, evaporate to dryness, reconstitute with 1 mL MeOH, inject a 10 jxL aliquot.
HPLC VARIABLES

Column: 200 X 4.6 5 jxm Silica 100-RP 18 Mobile phase: MeOH:water 50:50 Column temperature: 40 Flow rate: 1.5 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: k' 15.55, 18.13 (enantiomers)

OTHER SUBSTANCES

Also analyzed: bamethan, ephedrine, norpseudoephedrine, 1-phenylethylamine

KEYWORDS

derivatization; chiral
REFERENCE Vogt,C; Jira,T.; Beyrich,T. HPLC-Trennung racemischer Amine nach Derivatisierung mit (lS)-(+)-Campher-10-sulfonylchlorid, Pharmazie, 1990, 45, 691. SAMPLE

Matrix: bulk Sample preparation: Reflux a 10% excess of reagent in toluene for 10 min, add the drug, let stand at room temperature for 10 min, cool, dilute, inject an aliquot. (The reagent was N-(p-toluenesulfonyl)prolyl azide and was prepared as follows. Mix 40-45 mmoles L-(-)proline, 40 mL THF, and 200 mL 10% potassium carbonate, add 37-43 mmoles p-toluenesulfonyl chloride in 40 mL THF dropwise, heat at 50 and maintain at pH 8 or above

for 3 h, cool, acidify to pH 2, extract with chloroform. Extract the organic layers with potassium carbonate in water. Acidify the aqueous layer and extract it with chloroform. Dry the chloroform layer and evaporate it to dryness, recrystallize the resulting l-[(ptoluene)sulfonyl]proline from petroleum ether and benzene (Caution! Benzene is a carcinogen!) (Anal.Chem. 1984, 56, 958). Suspend 86 mmoles l-[(p-toluene)sulfonyl]proline in 15 mL water and add sufficient acetone to give a clear solution, cool to 0, add 10.2 g triethylamine in 175 mL acetone, slowly add 12.5 g ethyl chloroformate in 45 mL acetone while maintaining the temperature at 0, stir at 0 for 30 min, add dropwise 8.6 g sodium azide in 30 mL water, stir at 0 for 1 h, pour into ice water, extract with ether, dry over anhydrous magnesium sulfate, evaporate under reduced pressure at room temperature to give N-(p-toluenesulfonyl)prolyl azide (cf J.Org.Chem. 1961, 26, 3511).)
HPLC VARIABLES

Column: 300 X 4 7-9 jim silica gel Mobile phase: Petroleum ether:isopropanol 96.5:3.5 Flow rate: 1.5 Detector: UV 254
CHROMATOGRAM

Retention time: 13, 16 (enantiomers)

KEYWORDS

derivatization; chiral; normal phase

REFERENCE Zhou,Y.; Sun,Z.R; Lin,D.K. Liquid chromatographic evaluation of a new chiral derivatizing agent for enantiomeric resolution of amine and alcohol drugs, J.Liq.Chromatogr., 1990, 13, 875-885. SAMPLE

Matrix: bulk Sample preparation: Dissolve 10 ixmole compound (as free base or hydrochloride) in 500 IxL MeCN, add 250 |xL 5% sodium carbonate (for hydrochlorides only), add 500 JJLL 100 mM reagent in MeCN, vortex for 1 min, heat at 60 for 2 h, add 100 juimole L-proline, heat at 60 for 30 min. Remove a 100 \xL aliquot and dilute it with mobile phase, neutralize with acetic acid, inject a 10 jxL aliquot. Prepare the reagent ((R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL (lR,2R)-(-)-l,2-diaminocyclohexane, 12 mL water, and 12 mL EtOH, heat the oil bath to 80, add 2.8 mL carbon disulfide dropwise (making sure that the product does not start to precipitate), when addition is complete reflux for 1 h, acidify with 500 uX. 5 M HCl, reflux for 12 h, cool, filter, wash the solid with a little cold EtOH to give trans-4,5-tetramethyleneimidazolidine-2-thione as a white fluffy solid (mp 148-150) (Tetrahedron 1993, 49, 4419). Stir 7.97 g 3,5-dinitrobenzoyl chloride in 30 mL dichloroethane at 50, add a solution of 6 g trans-4,5-tetramethyleneimidazolidine-2-thione in 120 mL dichloroethane containing a catalytic amount of 4-(dimethylamino)pyridine over 15 min, reflux for 2 h, remove the crystals of (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate by filtration, evaporate the filtrate to dryness and dissolve the residue in 60 mL dichloroethane, reflux for 16 h to obtain more (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate (mp >250, Ia]546 = -133 (c = 1) in MeCN).
HPLC VARIABLES

Column: 125 X 4 5 juim Lichrospher 60 RP Select B Mobile phase: MeCN:20 mM ammonium acetate 45:55 Flow rate: 1 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: k' 11.86, k' 12.48 (enantiomers)

OTHER SUBSTANCES

Simultaneous: normetoprolol
KEYWORDS

derivatization; chiral
REFERENCE Kleidernigg,O.P.; Posch,K; Lindner,W. Synthesis and application of a new isothiocyanate as a chiral derivatizing agent for the indirect resolution of chiral amino alcohols and amines, J.Chromatogr.A, 1996, 729, 33-42. SAMPLE

Matrix: bulk, formulations Sample preparation: 10 mg Bulk drug, tablet, or capsule or 10 mL 1 mg/mL syrup + 5 mL 20% NaOH, sonicate until dissolved, add 10 mL 10 mM 2 naphthoyl chloride in dichloromethane, shake for 1 min. Remove the organic phase and wash the aqueous phase with 5 mL dichloromethane. Combine the organic layers and wash them with two 5 mL portions of 10 mM sulfuric acid, filter through a cotton plug, inject an aliquot.
HPLC VARIABLES

Guard column: 50 X 4.6 35-50 |xm silica Column: 250 X 4.6 Pirkle Covalent Phenylglycine (Regis) Mobile phase: Hexane:isopropanol:MeCN 97:3:0.5 Column temperature: 20 Flow rate: 2 Injection volume: 20 Detector: UV 280
CHROMATOGRAM

Retention time: k' 20 (-), k' 22 (+)

KEYWORDS

tablets; capsules; syrup; chiral; derivatization; 1% of minor enantiomer can be detected

REFERENCE WainerJ.W.; Doyle,T.D.; Adams,W.M. Liquid chromatographic chiral stationary phases in pharmaceutical analysis: determination of trace amounts of the (-)-enantiomer in (+)-amphetamine, J.Pharm.ScL, 1984, 73, 1162-1164. SAMPLE

Matrix: bulk, formulations Sample preparation: Bulk. Dissolve 10 mg bulk drug in 5 mL dichloromethane, add 5 mL 20% NaOH, add 10 mL 10 mM 2-naphthoyl chloride in dichloromethane, shake for 1 min. Remove the organic phase and wash the aqueous phase with 5 mL dichloromethane. Combine the organic layers and wash them with 5 mL 10 mM sulfuric acid. Filter the organic layer through a syringe containing a glass wool plug and anhydrous sodium sulfate, inject a 10 |xL aliquot. Capsules. Sonicate 1 capsule in 5 mL 20% NaOH for 45 min or until dissolved, filter, add 5 mL dichloromethane to filtrate, add 10 mL 10 mM 2naphthoyl chloride in dichloromethane, shake for 1 min. Remove the organic phase and wash the aqueous phase with 5 mL dichloromethane. Combine the organic layers and wash them with 5 mL 10 mM sulfuric acid. Filter the organic layer through a syringe containing a glass wool plug and anhydrous sodium sulfate, inject a 10 |xL aliquot.
HPLCVARIABLES

Column: Bakerbond Chiral Phase [DNBPG] Mobile phase: Hexane:isopropanol:MeCN 97:3:0.5 Column temperature: 20

Flow rate: 2 Injection volume: 10 Detector: UV 254

CHROMATOGRAM

Retention time: 28.5 (1), 31 (d)

KEY WORDS

capsules; chiral; derivatization

REFERENCE AlembikjM.C; Wainer,I.W. Resolution and analysis of enantiomers of amphetamines by liquid chromatography on a chiral stationary phase: collaborative study, J.Assoc.Off.Anal.Chem., 1988, 71, 530533. SAMPLE

Matrix: formulations Sample preparation: Powder tablet and add 50 mg to 50 mL MeCN:20 mM pH 3.8 phosphate buffer 3:97, sonicate for 5 min, filter (0.5 |xm), inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES

Guard column: Supelguard pre-column containing 5 |xm Suplex pKblOO (Supelco) Column: 150 X 4.6 5 fxm Suplex pKblOO (Supelco) Mobile phase: Gradient. MeCN:20 mM pH 3.8 phosphate buffer at 3:97 for 3 min, to 15: 85 over 5 min, stay at 15:85 for 4 min, re-equilibrate for 8 min. Flow rate: 1.5 Injection volume: 20 Detector: UV 220 for 5 min then UV 280
CHROMATOGRAM

Retention time: 3.62 Limit of quantitation: 10 jxg/mL

OTHER SUBSTANCES

Simultaneous: ephedrine, methamphetamine, caffeine, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, N-ethyl-3,4-methylenedioxyamphetamine

KEYWORDS

tablets
REFERENCE Longo,M.; Martines,C; Rolandi,L.; Cavallaro,A. Simple and fast determination of some phenethylamines in illicit tablets by base-activated reversed phase HPLC, J.Liq.Chromatogr., 1994, 27, 649-658. SAMPLE

Matrix: meconium Sample preparation: 500 mg Meconium + 5 mL water + 1 drop 500 mM HCl, vortex for 1 min, sonicate for 5 min, vortex for 1 min, centrifuge at 2683 g for 10 min. Remove the supernatant and add it to 5 mL water and 1 drop 500 mM HCl, vortex for 1 min, sonicate for 5 min, vortex for 1 min, centrifuge at 2683 g for 10 min. Make up the supernatant to 20 mL with pH 9.0 borax buffer, add it to an Extrelut SPE cartridge, after 10 min elute with 60 mL dichloromethanerisopropanol 80:20. Add 1 drop 2% tartaric acid in water and evaporate the eluate under a stream of nitrogen at 40, reconstitute the residue in 100 IxL mobile phase, inject a 20 |JLL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Supelcosil LC-18 DB

Mobile phase: MeOH:MeCN:THF:triethylamine:water 100:25:7:1.5:600 containing 77 mM KH2PO4 Flow rate: 1 Injection volume: 20 Detector: UV 204 CHROMATOGRAM Retention time: k' 4.3 Limit of detection: 500 ng/g OTHER SUBSTANCES Extracted: morphine Noninterfering: caffeine, benzoylecgonine, cocaine, codeine
KEY WORDS

SPE
REFERENCE Franssen,R.M.E.; Stolk,L.M.L.; van den Brand,W.; Smit,B.J. Analysis of morphine and amphetamine in meconium with immunoassay and HPLC-diode-array detection, JAnalToxicol., 1994, 18, 294-295. SAMPLE

Matrix: solutions Sample preparation: Mix 1 mL 20-300 |xg/mL amine solution in water with 2 mL 50 mg/ mL 4-nitrobenzoyl chloride in THF (freshly prepared) and 1 mL 1 M NaOH, heat at 65 for 1 h, cool, adjust pH to 12 with 1 M NaOH, extract with two 10 mL portions of chloroform. Combine the extracts and wash them with two 20 mL portions of 10% potassium carbonate, wash with water, dry over anhydrous magnesium sulfate. Evaporate to dryness under a stream of air, reconstitute the residue in MeOH, inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 jiBondapak C18 Mobile phase: MeCNrwater 35:65 Flow rate: 1.5 Injection volume: 5 Detector: UV 254 CHROMATOGRAM Retention time: 13
OTHER SUBSTANCES

Simultaneous: benzylamine, methamphetamine, a-methylbenzylamine, n-propylamphetamine

KEYWORDS

derivatization
REFERENCE Clark,R.C; Teague,J.D.; Wells,M.M.; Ellis,J.H. Gas and high-pressure liquid chromatographic properties of some 4-nitrobenzamides of amphetamines and related arylalkylamines, Anal.Chem., 1977, 49, 912-915. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 |xL aliquot.

HPLC VARIABLES

Column: 250 X 5 Spherisorb S5W Mobile phase: MeOH:buffer 90:10 (Buffer was 94 mL 35% ammonia and 21.5 mL 70% nitric acid in 884 mL water, adjust the pH to 10.1 with ammonia.) Flow rate: 2 Injection volume: 20 Detector: UV 254
CHROMATOGRAM Retention time: 2.62 OTHER SUBSTANCES

Simultaneous: morphine-N-oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine, pemoline, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phendimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphentermine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, tyramine, trimethoxyamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephedrine, dimethylamphetamine, methamphetamine, mescaline, mephentermine, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine, norpipanone, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamorphine, pentazocine, acetylcodeine, monoacetylmorphine Noninterfering: dopamine, levodopa, methyldopa, methyldopate, norepinephrine Interfering: norpseudoephedrine, fenfluramine, methylenedioxyamphetamine, thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine, ethylmorphine
REFERENCE Law,B.; Gill,R.; MofFat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of forensic interest on a silica column using an aqueous methanol eluent, J.Chromatogr., 1984, 301, 165-172. SAMPLE

Matrix: solutions Sample preparation: 50 jxL 1 mg/mL Compound in dichloromethane + 50 |xL 7.6 mM 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl isothiocyanate in MeCN or dichloromethane: triethylamine 99.8:0.2, mix, let stand at room temperature for 1 h, add 1 mL 1 M HCl, shake mechanically for 5 min, centrifuge at 1000 g for 15 min, discard the aqueous phase, add 1 mL 1 M NaOH, shake for 5 min, centrifuge at 1000 g for 15 min. Remove a 10 |xL aliquot of the organic layer and dilute it to 1 mL with MeOH, inject a 20-50 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.5 5 y,m octadecyl (IBM) Mobile phase: MeOH.water 55:45 Flow rate: 1-2 Injection volume: 20-50 Detector: UV 254
CHROMATOGRAM

Retention time: 16.8 (S), 18.2 (R) Limit of quantitation: 100 ng

OTHER SUBSTANCES

Simultaneous: l-(4-chlorophenyl)-2-aminopropane, l-(2,5-dimethoxy-4-methylphenyl)-2aminopropane, l-(2,4-dimethoxy-5-methylphenyl)-2-aminopropane, l-(2,5-dimethoxy-4thiomethylphenyl)-2-aminopropane, 1-phenylethylamine

KEY WORDS

derivatization; comparison with other derivatizing reagents; chiral

REFERENCE Miller,K.J.; Gal,J.; Ames,M.M. High-performance liquid chromatographic resolution of enantiomers of l-phenyl-2-aminopropanes (amphetamines) with four chiral reagents, J.Chromatogr., 1984, 307, 335-342. SAMPLE

Matrix: solutions Sample preparation: 100 jxL 1 mg/mL Compound in dichloromethane + 100 |xL 6.8 mM (R)-(+)-l-phenylethylisocyanate in dichloromethane, mix, let stand at room temperature for 1 h, evaporate to dryness under a stream of nitrogen, add 1 mL 100 mM NaOH, vortex for 15 min, add 1 mL 20% NaOH, add 3 mL dichloromethane, shake mechanically for 15 min, centrifuge at 1000 g for 15 min. Remove the organic layer and wash it with 2 mL 100 mM HCl. Remove a 100 JULL aliquot of the organic layer and dilute it to 1 mL with MeOH, inject a 20 JULL aliquot.
HPLC VARIABLES

Column: 250 X 4.5 5 \xm octadecyl (IBM) Mobile phase: MeOH.water 60:40 Flow rate: 1-2 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 13.5 (R), 14.0 (S) Limit of quantitation: 100 ng

OTHER SUBSTANCES

Simultaneous: l-(4-chlorophenyl)-2-aminopropane, l-(2,5-dimethoxy-4-methylphenyl)-2aminopropane, l-(2,4-dimethoxy-5-methylphenyl)-2-aminopropane, 1-phenylethylamine

KEY WORDS

derivatization; comparison with other derivatizing reagents; chiral

REFERENCE Miller,K.J.; Gal,J.; Ames,M.M. High-performance liquid chromatographic resolution of enantiomers of l-phenyl-2-aminopropanes (amphetamines) with four chiral reagents, J.Chromatogr., 1984, 307, 335-342. SAMPLE

Matrix: solutions Sample preparation: 2 mL THF + 1 mL 33.5 mM reagent in THF (freshly prepared) + 1 mL 1 mg/mL amphetamine in water + 700 |xL 10% sodium bicarbonate in water, heat at 65 for 1 h, cool, extract three times with 10 mL aliquots of chloroform. Combine the extracts and wash them with 10 mL water, dry over anhydrous magnesium sulfate, evaporate to dryness, reconstitute with 2.5 mL mobile phase, inject a 5 |xL aliquot. (Prepare reagent (l-[(4-nitrophenyl)sulfonyl]prolyl chloride) as follows. Mix 40-45 mmoles L-(-)-proline, 40 mL THF, and 200 mL 10% potassium carbonate, add 37-43 mmoles 4-nitrobenzenesulfonyl chloride in 40 mL THF dropwise, heat at 50 and maintain at pH 8 or above for 3 h, cool, acidify to pH 2, extract with chloroform. Extract the organic layers with potassium carbonate in water. Acidify the aqueous layer and extract it with chloroform. Dry the chloroform layer and evaporate it to dryness, recrystallize the resulting l-[(4nitrophenyl)sulfonyl]proline from petroleum ether and benzene (Caution! Benzene is a carcinogen!). Stir 15 mmoles l-[(4-nitrophenyl)sulfonyl]proline in 100 mL benzene and add 75 mmoles thionyl chloride in 50 mL benzene dropwise, heat at 35-40 until the

reaction is complete (about 48 h; monitor by IR), evaporate to dryness, recrystallize from n-heptane to give l-[(4-nitrophenyl)sulfonyl]prolyl chloride (Anal.Chem. 1984, 56, 958) (mp 110-110.5).)
HPLC VARIABLES

Guard column: 70 X 2.1 30-38 fim HC Pellosil (Whatman) Column: 150 X 4.6 5 |xm Supelcosil LC-Si Mobile phase: n-Heptane:chloroform 20:80 Flow rate: 1.4 Injection volume: 5 Detector: UV 254 CHROMATOGRAM Retention time: 4 (R), 4.5 (S)
KEYWORDS

derivatization; normal phase; chiral

REFERENCE Barksdale,J.M.; Clark,C.R. Liquid chromatographic determination of the enantiomeric composition of amphetamine and related drugs by diastereomeric derivatization, J.Chromatogr.Sci., 1985, 23, 176180. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 juig/mL, inject a 10 |ULL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 juim jxBondapak C18 Mobile phase: MeOH:acetic acid:triethylamine:water 15:1.5:0.5:83 Flow rate: 1.5 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 11
OTHER SUBSTANCES

Simultaneous: phenylpropanolamine, ephedrine, hydroxyamphetamine, methamphetamine, phentermine, mephentermine

REFERENCE Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE

Matrix: solutions Sample preparation: Dissolve 2 mg in 15 mL 0.45 M NaOH, extract twice with 30 mL chloroform. Combine the organic layers and add a 10% molar excess of 2,3,4,6-tetra-Oacetyl-p-D-glucopyranosyl isothiocyanate in chloroform, let stand for 10 min, evaporate the chloroform under a stream of air, dissolve the residue in 1 mL THF or MeOH, inject a 5 fiL aliquot.
HPLC VARIABLES

Guard column: 70 X 2.1 CorPell ODS

Column: 300 X 3.9 u-Bondapak C18 Mobile phase: MeOH:water:acetic acid 50:49:1 Flow rate: 1.5 Injection volume: 5 Detector: UV 254 CHROMATOGRAM Retention time: 25 (R), 28 (S)
OTHER SUBSTANCES

Simultaneous: norpseudoephedrine, norephedrine

KEY WORDS

chiral; derivatization
REFERENCE Noggle,F.T.,Jr.; DeRuiter,J.; Clark,C.R. Liquid chromatographic determination of the enantiomeric composition of amphetamine prepared from norephedrine and norpseudoephedrine, J.Chromatogr.ScL, 1987, 25, 38-42. SAMPLE

Matrix: solutions Sample preparation: Prepare a 500 |xg/mL solution in MeOH:water 50:50, inject a 5 u.L aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Zorbax C8 Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L MeCN:water 20:80. A:B from 100:0 to 0:100 over 30 min. (Purify triethylamine as follows. Wash neutral alumina (Merck) 3 times with 2 bed volumes of pentane, 3 times with 2 bed volumes of dichloromethane, and 3 times with 2 bed volumes of MeOH, allow solvent to evaporate in a fume hood overnight, heat alumina at 130 for 2 h. Prepare a 14 cm column of the washed alumina in a 290 X 22 tube, pass through a head volume of MeOH, pass through triethylamine. When triethylamine starts to elute discard the first 20 mL, use the next 20 mL, discard the column.) Flow rate: 2 Injection volume: 5 Detector: UV 210 CHROMATOGRAM Retention time: 9.2
OTHER SUBSTANCES

Simultaneous: acetophenone, desipramine, ethylmorphine, imipramine, mefenamic acid, methamphetamine, morphine, phenylbutazone, salicylic acid
KEY WORDS

also details of isocratic elution

REFERENCE Hill,D.W. Evaluation of alkyl bonded silica and solvent phase modifiers for the efficient elution of basic drugs on HPLC, J.Liq.Chromatogr., 1990, 13, 3147-3175.

SAMPLE Matrix: solutions

Sample preparation: Mix sample:50 mM (?) NaCN in 50 mM pH 9.3 borate buffer:25 (?) mM naphthalene-2,3-dicarboxaldehyde in MeOH 3:1:1, let stand for 15 min, inject a 50 |iL aliquot.
HPLC VARIABLES

Column: 200 X 3 5 jxm Chromspher ODS-2 C18 (Chrompack) Mobile phase: Gradient. A was THF:50 mM pH 6.8 potassium phosphate buffer 5:95. B was MeCN:MeOH:50 mM pH 6.8 potassium phosphate buffer 55:10:35. A:B from 70:30 to 0:100 over 1 h, maintain at 0:100 for 20 min. Flow rate: 0.5 Injection volume: 50 Detector: F ex 420 CHROMATOGRAM Retention time: 65
OTHER SUBSTANCES

Simultaneous: baclofen, tranylcypromine

KEYWORDS

derivatization
REFERENCE Koning,H.; Wolf,H.; Venema,K.; Korf,J. Automated precolumn derivatization of amino acids, small peptides, brain amines and drugs with primary amino groups for reversed-phase high-performance liquid chromatography using naphthalenedialdehyde as the fluorogenic label, J.Chromatogr., 1990, 533, 171-178. SAMPLE

Matrix: solutions Sample preparation: Mix 500 |xL of a solution in 20 mM pH 9.5 borate buffer with 100 IxL 10 mM NaCN in 20 mM pH 9.5 borate buffer, add 500 |xL 0.1 mM naphthalene-2,3dicarboxaldehyde in MeOH, mix, let stand at room temperature for 20 min, inject a 25 uL aliquot.
HPLC VARIABLES

Column: 150 X 3.1 5 urn LiChrosorb RP-18 Mobile phase: MeCN:2.5 mM pH 7.0 imidazole buffer 75:25 Flow rate: 0.5 Injection volume: 25 Detector: Chemiluminescence (418 nm cutoff filter) following post-column reaction. The column effluent mixed with 50 mM hydrogen peroxide in MeCN containing 5 mM bis(2nitrophenyl)oxalate pumped at 0.2 mL/min and the mixture flowed into the detector.
CHROMATOGRAM

Limit of detection: 4 fmole

KEYWORDS

derivatization
REFERENCE Kwakman,P.J.M.; Koelewijn,H.; Kool,L; Brinkman,U.A.T.; de Jong,G.J. Naphthalene- and anthraeene2,3-dialdehyde as precolumn labelling reagents for primary amines using reversed- and normal-phase liquid chromatography with peroxyoxalate chemiluminescence detection, J.Chromatogr., 1990, 511, 155-166. SAMPLE

Matrix: solutions

Sample preparation: Inject a 10 JJIL aliquot of a 100 ppm solution in MeCN:dichloromethane:triethylamine 50:50:0.05 into the mobile phase. The mobile phase flows through a 27 X 2.2 reactor packed with reagent at 72 to the column. (Reagent was dinitrobenzoyl-onitrobenzophenone polymeric reagent, prepared as follows. (Caution! Dioxane is carcinogenic in experimental animals! DMF may be carcinogenic! 3,5-Dinitrobenzoyl chloride and aluminum chloride are corrosive! Nitrobenzene is toxic!) Soxhlet extract 200-400 mesh polystyrene cross-linked with 4% divinylbenzene (Fluka) with MeCN for 48 h. Add 25 g aluminum trichloride in 300 mL dry nitrobenzene to a mixture of 50 g of the polystyrene resin and 100 g 4-chloro-3-nitrobenzoyl chloride, stir the mixture mechanically at 60 for 5 h, pour into a mixture of 150 mL DMF, 100 mL concentrated HCl, and 150 g ice. Wash the beads with 300 mL portions of DMF:water 75:25 until the washings are colorless, wash with warm DMF (60), wash with six 300 mL portions of dichloromethane:MeOH 2:1, dry. Add the polymer to 130 mL 40% benzyltrimethylammonium hydroxide in water, 130 mL water, and 260 mL dioxane, heat at 90 for 8 h, filter, repeat the process, filter, wash the beads with four portions of warm (60) dioxane, add 30 mL acetic acid, stir for 15 min, wash with dioxane until the washings are neutral, wash with six 300 mL portions of dichloromethane:MeOH 2:1. Add a portion of polymer to dry chloroform, add a threefold excess of 3,5-dinitrobenzoyl chloride and pyridine, stir at 0-10 for 30 min, filter off polymer, wash with chloroform to give the reagent (J.Org.Chem. 1984, 49, 922).)
HPLC VARIABLES

Column: 250 X 4.6 5 jxm LC-(R)-Naphthyl Urea (Supelco) Mobile phase: Hexane:dichloromethane:THF 70:27:3 Flow rate: 0.1 for 40 s, to 3.1 over 30 s, maintain at 3.1 Injection volume: 10 Detector: UV
CHROMATOGRAM

Retention time: 11.5 (D), 13 (L)

KEYWORDS

derivatization; chiral
REFERENCE Bourque,A.J.; KrullJ.S. Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection, J.Chromatogr., 1991, 537, 123-152.

SAMPLE Matrix: solutions

HPLC VARIABLES

Column: 150 X 4.6 Supelcosil LC-ABZ Mobile phase: MeCN:25 mM pH 6.9 potassium phosphate buffer 35:65 Flow rate: 1.5 Injection volume: 25 Detector: UV 254 CHROMATOGRAM Retention time: 2.220
OTHER SUBSTANCES

Also analyzed: 6-acetylmorphine, amiloride, benzocaine, benzoylecgonine, caffeine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine, levorphanol, LSD, meperidine, mephobarbital, methadone, methylphenidate, methyprylon, Nnorcodeine, oxazepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine

REFERENCE Ascah,T.L. Improved separations of alkaloid drugs and other substances of abuse using Supelcosil LCABZ column, Supelco Reporter, 1993,12(3), 18-21. SAMPLE

Matrix: solutions Sample preparation: Mix 50 JXL of a 200 ppm solution in MeCN:500 mM pH 9.0 borate buffer 50:50 with 25 mg reagent, after 1 min elute with 1 mL hexane:THF 75:25, inject a 5 |xL aliquot. (Reagent was dinitrophenyl carbamate benzotriazole polymeric reagent, synthesized as follows. (Caution! Chloroform, dichloromethane, dioxane, and hydrazine are carcinogenic in experimental animals! DMF may be carcinogenic! 3,5-dinitrobenzoyl chloride and aluminum chloride are corrosive! Nitrobenzene is toxic!) 10 g Dried macroporous polystyrene (Xe-305, Rohm and Haas) + 1Og 3-nitro-4-chlorobenzyl alcohol + 10 g anhydrous aluminum chloride + 50 mL nitrobenzene, heat at 65-70 for 3 days, cool, filter, wash polymer with three 50 mL portions of 1 M HCl in dioxane, with three 50 mL portions of DMF, with three 50 mL portions of MeOH, and with three 50 mL portions of dichloromethane, dry under vacuum at 100. Reflux 19 g of this polymer in 60 mL hydrazine hydrate:ethylene glycol monoethyl ether 40:60 for 20 h, cool to room temperature, filter off the polymer and wash it thoroughly with water. Suspend the polymer in 100 mL concentrated HCl:dioxane 50:50, reflux for 20 h, filter the polymer and wash it with five 100 mL portions of water, with three 100 mL portions of MeOH, and with three 50 mL portions of ether, dry under vacuum at 80. Functionalization was 1.17 mmoles/g (Eur.J.Biochem. 1975, 59, 55). Dissolve 3,5-dinitrobenzoyl chloride in the minimum amount of glacial acetic acid, add an equimolar amount of sodium azide, stir for 30 min, dilute with water, filter to obtain 3,5-dinitrobenzoyl azide (Caution! Azides are toxic and potentially explosive!) (J. Liq. Chromatogr. 1986, 9, 443). Heat 71 mg 3,5-dinitrobenzoyl azide in 15 mL toluene (dried over calcium hydride) at ??? for 30 min, cool using an ice bath, add 200 mg polymer, allow to warm to room temperature with stirring for 1 h, filter, wash the polymer with four 10 mL portions of warm (40) dichloromethane, dry under high vacuum for I h . )
HPLC VARIABLES

Column: 250 X 4.6 5 fim LC-(R)-naphthylurea (Supelco) Mobile phase: Hexane:EtOH:MeCN 93:7:0.5 Flow rate: 2 Injection volume: 5 Detector: UV 254
CHROMATOGRAM

Retention time: 12.2, 15.7 (enantiomers)

OTHER SUBSTANCES

Simultaneous: methamphetamine
KEYWORDS

derivatization; chiral
REFERENCE Bourque,A.J.; KrullJ.S. Immobilized isocyanates for derivatization of amines for chiral recognition in liquid chromatography with UV detection, J.Pharm.Biomed.AnaL, 1993, 11, 495-503. SAMPLE

Matrix: solutions Sample preparation: 50 JULL 5 mg/mL Amphetamine in 100 mM HCl + 50 |xL buffer + 100 IxL reagent, swirl for 1 min, place on ice for 5 min, add 2 mL mobile phase, inject a 5 JJLL aliquot. (Buffer was 100 mM sodium borate adjusted to pH 9.50 with 2 M NaOH. Reagent was 13.40 g o-phthaldialdehyde and 21.8 mg 1-thio-p-D-glucose in 1 mL MeOH, protect from light, keep on ice.)

HPLC VARIABLES

Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: MeOH:MeCN:buffer 55:2:45 (Buffer was 3 mL/L glacial acetic acid in water, pH adjusted to 7.20 with 2 M NaOH.) Flow rate: 1 Injection volume: 5 Detector: F ex 338 em 425 or UV 254
CHROMATOGRAM

Retention time: 12.37 (R-(-)), 14.12 (S-(+))

KEYWORDS

derivatization; protect from light; chiral

REFERENCE Desai,D.M.; Gal,J. Enantiospecific drug analysis via the orffto-phthalaldehyde/homochiral thiol derivatization method, J.Chromatogr., 1993, 629, 215-228. SAMPLE

Matrix: solutions
HPLC VARIABLES

Guard column: 30 X 2.1 Spheri-5 RP-8 Column: 220 X 2.1 Spheri-5 RP-8 Mobile phase: Gradient. A was 0.08% diethylamine and 0.09% phosphoric acid in water, pH 2.3. B was MeCNrwater 90:10 containing 0.08% diethylamine and 0.09% phosphoric acid. A:B 95:5 for 2 min, to 0:100 over 15 min (?), maintain at 0:100 for 5 min. Column temperature: 50 Flow rate: 0.5 Detector: UV 200 CHROMATOGRAM Retention time: 6.5
OTHER SUBSTANCES

Simultaneous: diethylpropion, phenylpropanolamine, ephedrine, methamphetamine, phentermine, fenfluramine Also analyzed: amitriptyline, chlordiazepoxide, chlorpromazine, desalkymurazepam, desipramine, desmethyldoxepin, diazepam, doxepin, flurazepam, imipramine, mesoridazine, norchlordiazepoxide, nordiazepam, nortriptyline, oxazepam, prazepam, promazine, thioridazine, thiothixene, trifluoperazine
REFERENCE Rainin Catalog, Cl-94, 1994, p. 7.24. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210

OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazOlamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phalse solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994, 18, 233-242. SAMPLE

Matrix: solutions Sample preparation: Mix a 100 |xL aliquot of a 0.1-1 mM solution in 10 mM HCl with 500 fxL 100 mM pH 9.5 borate buffer, 100 |xL 2.5 mM N-acetyl-L-cysteine in 10 mM HCl, and 200 JJIL 5 mM o-phthalaldehyde in EtOH, let stand for 10 min, inject an aliquot.

HPLC VARIABLES

Column: 125 X 4 5 |xm LiChrospher 100 RP-18 end-capped Mobile phase: MeOH:50 mM pH 6.5 phosphate buffer 60:40 Flow rate: 1 Injection volume: 20 Detector: UV 335
CHROMATOGRAM

Retention time: k' 3.5 (a = 1.13, R8 = 1.04)

KEYWORDS

derivatization; chiral; comparison with capillary electrophoresis; comparison with other derivatizing reagents
REFERENCE Leroy,R; Bellucci,L.; Nicolas,A. Chiral derivatization for separation of racemic amino and thiol drugs by liquid chromatography and capillary electrophoresis, Chirality, 1995, 7, 235-242. SAMPLE

Matrix: solutions Sample preparation: 1 mL Solution + 500 |xL 0.5% sodium l,2-naphthoquinone-4-sulfonate in water + 500 JJLL buffer, let stand for 10 min, extract three times with 2 mL aliquots of n-hexane:ethyl acetate 50:50. Combine the organic layers and evaporate them to dryness at 80, reconstitute with 2 mL MeCNrwater 50:50, inject a 50 |JLL aliquot. (Buffer was 4% sodium bicarbonate adjusted to pH 10 with 10% NaOH.)
HPLC VARIABLES

Column: 250 X 4 5 fxm Hypersil ODS C18 Mobile phase: Gradient. MeCN:0.5% propylamine hydrochloride in water from 40:60 to 50: 50 over 2.5 min, to 70:30 over 1 min, maintain at 70:30 for 4.5 min. Flow rate: 1 Injection volume: 50 Detector: UV 280 CHROMATOGRAM Retention time: 5 OTHER SUBSTANCES Extracted: methamphetamine
KEY WORDS

derivatization
REFERENCE Herraez-Hernandez,R.; Campins-Falc6,R; Sevillano-Cabeza,A. On-line derivatization into precolumns for the determination of drugs by liquid chromatography and column switching: Determination of amphetamines in urine, Anal.Chem., 1996, 68, 734-739. SAMPLE

Matrix: solutions, urine Sample preparation: Add 20 |xL of a solution of amphetamine in MeCNiwater 50:50 to a cartridge containing 30 mg reagent, heat at 60 for 5 min, elute with 500 |xL MeCN, inject a 20 |xL aliquot of the eluate. Alternatively, inject 10 jxL of urine (adjusted to pH 10 with 100 mM NaOH and filtered) into a 27 X 3 reactor filled with reagent held at 60, after 5 min elute the contents of the reactor on to the column with mobile phase, elute the column with mobile phase, monitor the effluent from the column. (Prepare reagent as follows. Add 3 g aluminum trichloride to 5 g 60-90 |xm macroporous polystyrene-divinylbenzene

copolymer (Fluka) and 10 g 4-chloro-3-nitrobenzoyl chloride stirred in 100 mL dry nitrobenzene, stir at 60 for 5 h, pour into a mixture of 75 mL DMF, 50 mL concentrated HCl, and 50 g ice, filter. Wash the solid with three 50 mL portions of DMF:water 75:25, wash with 30 mL warm (60) DMF, wash with five 50 mL portions of dichloromethane:MeOH 2:1. Stir the product in 15 mL 40% benzyltrimethylammonium hydroxide in water, 15 mL water, and 30 mL dioxane (Caution! Dioxane is a carcinogen!) at 90 for 8 h, filter. Wash the product with four 50 mL portions of warm (60) dioxane. Stir the solid with 30 mL acetic acid for 15 min, filter. Wash the solid with three 50 mL portions of dioxane until the washings are neutral, wash with four 50 mL portions of dichloromethane:MeOH 2:1. Stir 1 g of the yellow product in 10 mL dichloromethane and 500 |xL pyridine, add 1.24 g 9-fluorenylmethyl chloroformate, stir at room temperature for 30 min, filter. Wash the solid with three 20 mL portions of DMF, three 20 mL portions of dichloromethane, and three 20 mL portions of dry diethyl ether, dry under vacuum to give the reagent (polymer bound o-nitrobenzophenone fluorenylmethyl carbonate) Store in the freezer, good for at least a year.)
HPLC VARIABLES

Column: 250 X 4.6 5 ^m LiChroSpher C18 Mobile phase: MeCN:water 50:50 Flow rate: 1.5 Injection volume: 20 Detector: F ex 265 em 320
CHROMATOGRAM

Retention time: 5 Limit of detection: 100 ppb

OTHER SUBSTANCES

Also analyzed: butylamine, diethylamine, morpholine, propylamine

KEY WORDS

derivatization
REFERENCE Gao,C.-X.; Chou,T.-Y; KrullJ.S. Polymeric activated ester reagents for off-line and on-line derivatizations of amine nucleophiles in high-performance liquid chromatography with ultraviolet and fluorescence detection, Anal. Chem., 1989, 61, 1538-1548. SAMPLE

Matrix: urine Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH, 3 mL MeCN: 10 mM ammonium acetate 40:60 adjusted to pH 3 with acetic acid, and 5 mL water. 5 mL Urine + 5 mL 500 mM ammonium acetate, adjusted to pH 9.5 with ammonia, mix, add to the SPE cartridge, wash with 20 mL 5 mM pH 9.5 ammonium acetate, wash with 0.5 mL water. Elute with 2 mL MeCN: 10 mM ammonium acetate 40:60 adjusted to pH 3 with acetic acid, inject a 50 jxL aliquot of the eluate.
HPLC VARIABLES

Column: 150 X 4.6 L-column ODS (Chemical Inspection & Testing Institute, Tokyo) Mobile phase: Gradient. MeCN: 100 mM ammonium acetate 0:100 for 1 min, to 40:60 over 20 min. Flow rate: 1 Injection volume: 50 Detector: UV 210; MS Shimadzu model QP-1100EX thermospray, vaporizer temperature from 170 to 150 over 20 min. SIM, m/z 136
CHROMATOGRAM
Retention time: 14.7

Limit of detection: 2-40 ng/mL

OTHER SUBSTANCES

Extracted: 6-acetylmorphine, benzoylecgonine, cocaine, ephedrine, methamphetamine, methylephedrine, morphine, morphine-3-glucuronide, morphine-6-glucuronide
KEYWORDS

SPE
REFERENCE Tatsuno,M.; Nishikawa,M.; Katagi,M.; Tsuchihashi,H. Simultaneous determination of illicit drugs in human urine by liquid chromatography-mass spectrometry, JAnal.ToxicoL, 1996, 20, 281-286. SAMPLE

Matrix: urine Sample preparation: 500 (xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 |xL buffer, centrifuge at 11000 g for 30 s, inject a 500 jxL aliquot onto column A with mobile phase A, after 0.6 min backflush column A with mobile phase A to waste for 1.6 min, elute column A with 250 |iL mobile phase B, with 200 uL mobile phase C, and with 1.15 mL mobile phase D. Elute column A to waste until drugs start to emerge then elute onto column B. Elute column B to waste until drugs started to emerge, then elute onto column C. When all the drugs have emerged from column B remove it from the circuit, elute column C with mobile phase D, monitor the effluent from column C. Flush column A with 7 mL mobile phase E, with mobile phase D, and mobile phase A. Flush column B with 5 mL mobile phase E then with mobile phase D. (Buffer was 6 M ammonium acetate adjusted to pH 8.0 with 2 M KOH.)
HPLC VARIABLES

Column: A 10 X 2.1 12-20 |xm PRP-I spherical poly(styrene-divinylbenzene) (Hamilton); B 10 x 3.2 11 |xm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 |jim C8 (Phenomenex) + 150 X 4.6 5 |xm silica (Macherey-Nagel) Mobile phase: A 0.1% pH 8.0 potassium borate buffer; B 6 mM KH2PO4 containing 5 mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid; C MeCN:buffer 40:60 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.); D MeCN.buffer 33:67 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.); E MeCN:buffer 70:30 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.) Column temperature: ambient (column A), 40 (columns B and C) Flow rate: A 5; B-E 1 Injection volume: 500 Detector: UV 210, UV 235
CHROMATOGRAM Retention time: k' 2.5

Internal standard: N-ethylnordiazepam (k' 2.1), chlorpheniramine (k' 5.9) Limit of detection: 300 ng/mL
OTHER SUBSTANCES

Extracted: methamphetamine, desipramine, nortriptyline, diphenhydramine, methadone, imipramine, flurazepam, amitriptyline, morphine, codeine, hydromorphone, hydrocodone, caffeine, cotinine, benzoylecgonine, secobarbital, oxazepam, phenobarbital, nordiazepam, diazepam Interfering: phenylpropanolamine, phentermine, phenmetrazine, lidocaine, ephedrine, pentazocine

KEYWORDS

column-switching
REFERENCE Binder,S.R.; Regalia,M.; Biaggi-McEachern,M.; Mazhar,M. Automated liquid chromatographic analysis of drugs in urine by on-line sample cleanup and isocratic multi-column separation, J.Chromatogr., 1989, 473, 325-341. SAMPLE

Matrix: urine Sample preparation: Adjust pH of urine to 10 with 1 M NaOH, inject a 10 (xL aliquot onto column A heated to 60, after 6 s stop the flow through column A, after 5 min backflush the contents of column A onto column B with mobile phase, elute column B with mobile phase, monitor the effluent from column B.
HPLC VARIABLES

Column: A 27 X 30 packed with polymeric reagent; B 250 X 4 5 jim LiChrospher C18 (Prepare polymeric reagent, polymeric FMOC-L-proline, as follows. Add 25 g aluminum trichloride in 300 mL dry nitrobenzene to 50 g 60-90 |xm 96% styrene-4% divinylbenzene resin (Fluka) and 100 g 4-chloro-3-nitrobenzoyl chloride, stir mechanically at 60 for 5 h, pour into a mixture of 150 mL DMF, 100 mL concentrated HCl, and 150 g ice, filter. Wash the solid with 300 mL portions of DMF:water 75:25 until the washings are colorless, wash with warm (60) DMF, wash with six 300 mL portions of dichloromethane:MeOH 2:1. Stir the product in 130 mL 40% benzyltrimethylammonium hydroxide in water, 130 mL water, and 260 mL dioxane at 90 for 8 h, filter, repeat the process. Wash the product with four portions of warm (60) dioxane. Stir the solid with 30 mL acetic acid for 15 min, filter. Wash the solid with dioxane until the washings are neutral, wash with six 300 mL portions of dichloromethane:MeOH 2:1 to give a nitrobenzophenol-substituted polymer (J. Org. Chem. 1984, 49, 924). Dissolve 600 mg N-(9-fluorenylmethoxycarbonyl)-L-proline in 10 mL dichloromethane, cool to 0, add 1.8 mmoles dicyclohexylcarbodiimide, stir at 0 for 30 min, filter. Add the filtrate to 1 g resin, add 500 |xL pyridine, stir at room temperature for 1 h, filter, wash with three 50 mL portions of hexane, wash with three 50 mL portions of dichloromethane, wash with three 100 mL portions of MeCN.) Mobile phase: MeCN:water 48:52 Flow rate: 1.5 Injection volume: 10 Detector: UV 265, F ex 265 em 315 CHROMATOGRAM Retention time: 28 (d), 30 (1) Limit of detection: 50 ng/mL
KEYWORDS

derivatization; chiral; column-switching

REFERENCE Gao,C.-X.; KrullJ.S. Determination of enantiomeric drugs in physiological fluids using on-line solid phase derivatizations and reversed-phase liquid chromatography, J.Pharm.Biomed.Anal., 1989, 7, 1183-1198. SAMPLE

Matrix: urine Sample preparation: 500 jxL Urine + 100 |xL 25 |xg/mL N-n-propylaniline + 6 mL pH 10.0 carbonate buffer + 15 mL water, add mixture to an Extrelut SPE cartridge, let stand for 20 min, elute with 40 mL hexane:ethyl acetate 90:10. Add the eluate to 3 mL 100 mM sulfuric acid and 500 mg NaCl, stir for 20 min, centrifuge at 1000 g for 5 min. Remove the lower layer and add it to 3 mL 2.5 M NaOH and 20 JJLL benzoyl chloride, stir vigorously

for 30 min. Extract the mixture with 1.5 mL chloroform. Wash the chloroform layer twice with 5 mL water and evaporate it to dryness at 40, reconstitute the residue in 200 |JLL hexane:isopropanol 90:10, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Chiralcel OB + 250 X 4.6 Chiralcel OJ Mobile phase: Hexane:isopropanol 90:10 Column temperature: 48 Flow rate: 1-1.4 Detector: UV 220 CHROMATOGRAM Retention time: 17 (1), 25 (d) Internal standard: N-n-propylaniline (11) Limit of detection: 25 ng OTHER SUBSTANCES Extracted: methamphetamine
KEYWORDS

rat; SPE; derivatization; chiral

REFERENCE Nagai,T.; Kamiyama,S. Assay of the optical isomers of methamphetamine and amphetamine in rat urine using high-performance liquid chromatography with chiral cellulose-based columns, J.Chromatogr., 1990, 525, 203-209. SAMPLE

Matrix: urine Sample preparation: Mix 100 u-L urine with 60 mg reagent, after 2 min elute with 500 |iL MeCN, add 500 |xL 50 mM NaOH to the eluate, mix, inject a 20 |xL aliquot. (The reagent was dinitrobenzoylbenzotriazole polymeric reagent, synthesized as follows. (Caution! Chloroform, dichloromethane, dioxane, and hydrazine are carcinogenic in experimental animals! DMF may be carcinogenic! 3,5-Dinitrobenzoyl chloride and aluminum chloride are corrosive! Nitrobenzene is toxic!) 10 g Dried macroporous polystyrene (Xe305, Rohm and Haas) + 1Og 3-nitro-4-chlorobenzyl alcohol +1Og anhydrous aluminum chloride + 50 mL nitrobenzene, heat at 65-70 for 3 days, cool, filter, wash polymer with three 50 mL portions of 1 M HCl in dioxane, with three 50 mL portions of DMF, with three 50 mL portions of MeOH, and with three 50 mL portions of dichloromethane, dry under vacuum at 100. Reflux 19 g of this polymer in 60 mL hydrazine hydrate:ethylene glycol monoethyl ether 40:60 for 20 h, cool to room temperature, filter off the polymer and wash it thoroughly with water. Suspend the polymer in 100 mL concentrated HCl: dioxane 50:50, reflux for 20 h, filter the polymer and wash it with five 100 mL portions of water, with three 100 mL portions of MeOH, and with three 50 mL portions of ether, dry under vacuum at 80. Functionalization was 1.17 mmoles/g (Eur.J.Biochem. 1975, 59, 55). Add a portion of polymer to dry chloroform, add a three-fold excess of 3,5-dinitrobenzoyl chloride and pyridine, stir at 0-10 for 30 min, filter off polymer, wash with chloroform to give the reagent (J.Org.Chem. 1984, 49, 922).)
HPLC VARIABLES

Column: 125 X 4 5 fim LiChrosorb C18 Mobile phase: MeCN:water 50:50 containing 0.05% ammonium hydroxide Flow rate: 2 Injection volume: 20 Detector: UV CHROMATOGRAM Retention time: 4.1

KEYWORDS

derivatization
REFERENCE Bourque,A.J.; KrullJ.S. Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection, J.Chromatogr., 1991, 537, 123-152. SAMPLE

Matrix: urine Sample preparation: Inject a 10 |JLL aliquot into a 27 X 2 reactor packed with 65 mg polymeric reagents A and B in a 5:1 ratio heated at 60, after 10 min flush the contents of the reactor onto the column with the mobile phase. (Prepare the polymeric reagents as follows. Stir mechanically 5 g 60-90 jxm polystyrene-divinylbenzene copolymer (Fluka), 10 g 4-chloro-3-nitrobenzoyl chloride, and 3 g aluminum trichloride in 100 mL dry nitrobenzene at 60 for 5 h, pour into a mixture of 75 mL DMF, 50 mL concentrated HCl, and 50 g ice, filter. Wash the solid with three 50 mL portions of DMF:water 75:25, wash with warm (60) DMF, wash with three 50 mL portions of dichloromethane:MeOH 2:1. Add the solid to 15 mL 40% benzyltrimethylammonium hydroxide in water, 15 mL water, and 30 mL dioxane (Caution! Dioxane is a carcinogen!), heat at 90 for 8 h, filter, wash with four 50 mL portions of warm (60) dioxane. Add 30 mL acetic acid:water 50:50 and stir for 15 min, filter, wash with water until the washings are neutral, wash the three 50 mL portions of dioxane, wash with four 50 mL portions of dichloromethane:MeOH 2:1. Stir 1.12 g of this product with 248 mg 9-fluorenylmethyl chloroformate in 2 mL dichloromethane and 100 jxL pyridine at room temperature for 30 min, filter, wash with three 4 mL portions of DMF, wash with three 4 mL portions of dichloromethane, wash with three 4 mL portions of dry ethyl ether, dry under vacuum to give polymer-bound fluorenylmethoxycarbonyl reagent (A). Substitute 4-nitrobenzoyl chloride for 9-fluorenylmethyl chloroformate to obtain polymer-bound 4-nitrobenzoyl reagent (B).)
HPLC VARIABLES

Column: 250 X 4 5 |xm LiChrospher C18 Mobile phase: MeCN:water 55:45 Flow rate: 1.5 for 6 min then 2.5 Injection volume: 10 Detector: UV 265, F ex 265 em 320
CHROMATOGRAM

Retention time: 6 (4-nitrobenzoyl derivative), 15 (fluorenylmethoxycarbonyl derivative) Limit of quantitation: 10 ppm (F)
KEYWORDS

derivatization
REFERENCE Gao,C.X.; Schmalzing,D.; KrullJ.S. A mixed-bed, multi-derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection, Biomed.Chromatogr., 1991, 5, 23-31. SAMPLE

Matrix: urine Sample preparation: 200-500 |xL Rat urine + 200-500 fxL pH 3.8 acetate buffer + 25 JLJLL 40 fig/mL p-glucuronidase and 20 u,g/mL arylsulfatase (Merck), heat at 37 for 24 h, add 100 |xL 25 |uLg/mL 3-methoxytyramine in water, add 100 u.L water, adjust pH to 9.0 with 1.9 M sodium carbonate, add to an Extrelut SPE cartridge, let stand for 20 min, elute with 6 mL ethyl acetate. Add the eluate to 1 mL 100 mM sulfuric acid, extract. Add the aqueous layer to 3 mL 2.5 M NaOH, add 25 |xL benzoyl chloride, extract with 5 mL ethyl

acetate. Wash the ethyl acetate layer with water, evaporate to dryness under a stream of nitrogen at 40, reconstitute the residue in 50 jxL EtOH, 3.5 mL 50 mM pH 8.0 Tris/HCl buffer, and 35 |xL esterase (Type 1 porcine liver, Sigma). Heat at 25 for 45 min, add to an activated Sep-Pak C18 SPE cartridge, wash with 5 mL water, elute with 5 mL acetone. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL hexane:EtOH 89:11, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Chiralcel OB + 250 X 4.6 Chiralcel OJ Mobile phase: n-Hexane:EtOH 89:11 Column temperature: 48 Flow rate: 1.4 Injection volume: 20 Detector: UV 220
CHROMATOGRAM

Retention time: 12 (L), 14 (D) Internal standard: 3-methoxytyramine (54)

OTHER SUBSTANCES

Extracted: metabolites, methamphetamine

KEYWORDS

rat; SPE; derivatization

REFERENCE Nagai,T.; Kamiyama,S. Simultaneous HPLC analysis of optical isomers of methamphetamine and its metabolites, and stereoselective metabolism of racemic methamphetamine in rat urine, JAnaLToxicoL, 1991, 15, 299-304. SAMPLE

Matrix: urine Sample preparation: Adjust pH of 3 mL urine to 11 with 10 M KOH, add to an Extrelut 3 column, let stand for 10 min, elute with 15 mL n-hexane into a tube containing one drop of 3 M HCl. Evaporate the eluate to dryness under a stream of nitrogen at 35. Add 1.5 mL 8% sodium bicarbonate in water and 1 mL 0.5% sodium naphthoquinone-4-sulfonate in water to the residue, heat at 70 for 20 min, cool, extract with 5 mL carbon tetrachloride. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |xL mobile phase, inject a 20 |JLL aliquot.
HPLC VARIABLES

Guard column: 4 x 4 5 jjim Lichrospher 100 RP8 Column: 250 X 4 5 |xm Lichrospher 100 RP8 Mobile phase: MeCN:buffer 55:45 (Buffer was 1.361 g KH2PO4 in 950 mL, add 1.3 mL methanesulfonic acid, adjust pH to 3 with 5 M KOH, make up to 1 L with water.) Flow rate: 1 Injection volume: 20 Detector: UV 460
CHROMATOGRAM

Retention time: 7.3 Limit of detection: 60 ng/mL

OTHER SUBSTANCES

Extracted: methamphetamine Noninterfering: acetaminophen, aspirin, amitriptyline, buprenorphine, caffeine, carbamazepine, chlorpromazine, desipramine, dextromethorphan, doxepin, ephedrine, fenfluramine, imipramine, lidocaine, loxapine, meperidine, methadone, methaqualone, naloxone,

naltrexone, nicotine, orphenadrine, oxycodone, papaverine, pentazocine, phendimetrazine, phenmetrazine, phentermine, phenylpropanolamine, phenytoin, primidone, procaine, promethazine, propoxyphene, propyphenazone, theobromine, theophylline, trazodone, triflupromazine, trimethoprim, trimipramine
KEYWORDS

SPE; derivatization
REFERENCE Ferrara,S.D.; Tedeschi,L.; Frison,G.; Castagna,F. Solid-phase extraction and HPLC-UV confirmation of drugs of abuse in urine, JAnal.ToxicoL, 1992, 16, 217-222. SAMPLE

Matrix: urine Sample preparation: Condition a 100 mg Adsorbex SCX cation-exchange SPE cartridge (Merck) with 2 mL MeOH, 1 mL water, and 1 mL 17 mM KH2PO4, do not allow to dry. Centrifuge urine at 2000 g for 5 min. 1 mL Urine + 500 |xL 50 mM KH2PO4, sonicate for 1 min, add to the SPE cartridge, rinse vial with 50 JJLL 50 mM KH2PO4 and add to cartridge, dry cartridge for 1 min, wash with three 500 |xL portions of 17 mM KH2PO4, wash with 1 mL MeOH, dry under vacuum for 1 min, elute with four 500 |xL portions of MeOH: 7.3% HCl (97.5:2.5) at a flow rate of 0.5 mIVmin, inject a 10 (LJLL aliquot.
HPLC VARIABLES

Column: 125 X 4 3 jjim Spherisorb ODS-I Mobile phase: Gradient. A was water containing 5 mL (8.5 g) 85% orthophosphoric acid and 280 |xL (0.22 g) hexylamine per liter. B was MeCN containing 100 mL water, 5 mL (8.5 g) 85% orthophosphoric acid, and 280 jxL (0.22 g) hexylamine per liter. A:B 94.5:5.5 for 10.6 min, then to 61:39 over 11 min. Column temperature: 40 Flow rate: 0.8 Injection volume: 10 Detector: UV 198
CHROMATOGRAM

Retention time: 6 Limit of detection: 30 ng/mL

OTHER SUBSTANCES

Extracted: 3,4-methylenedioxyamphetamine, methamphetamine, 4-methoxyamphetamine, phentermine, 3,4-methylenedioxymethamphetamine, 5-methoxy-3,4-methylenedioxyamphetamine, 3,4,5-trimethoxyamphetamine, 3,4-methylenedioxyethylamphetamine, 2,5dimethoxyamphetamine, 4-bromo-2,5-dimethoxyphenylethylamine, 2,5-dimethoxy-4methylamphetamine, 4-bromo-2,5-dimethoxyamphetamine, 2,5-dimethoxy-4-ethylamphetamine, mescaline, methoxamine
KEY WORDS

SPE
REFERENCE Helmlin,H.-J.; Brenneisen,R. Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection, J.Chromatogr., 1992, 593, 87-94.

SAMPLE Matrix: urine Sample preparation: Condition a 130 mg Bond Elut Certify SPE cartridge with 3 mL MeOH and 3 mL 100 mM pH 6.0 phosphate buffer, do not allow to go dry. 2 mL Urine +

800 jxL 100 mM pH 6.0 phosphate buffer + 200 |xL 10 jxg/mL l-methyl-3-phenylpropylamine in MeOH, if necessary adjust pH to 5-7 with 1 M KOH or 1 M HCl (with pH paper), add the mixture to the SPE cartridge, wash with 1 mL 1 M acetic acid, wash with 3 mL water, dry for 5 min under vacuum, wash with 6 mL MeOH, dry for 2 min under vacuum, elute with 2 mL ethyl acetate:30% ammonium hydroxide 98:2 at a flow rate of 4-6 drops/ sec. Evaporate the eluate for 2 min under a stream of nitrogen, add 100 |xL 1 M HCl in diethyl ether, evaporate to dryness under a stream of nitrogen at 40, reconstitute the residue in 125 |xL MeCN:water:triethylamine 90:8.6:1.4. Add the entire volume to 50 5 mg reagent in a 1 mL plastic pipette tip plugged with a Kimwipe, react for 30 s, elute with 500 JJLL MeCN under pressure, inject a 10-20 |xL aliquot. (The reagent was dinitrobenzoylbenzotriazole polymeric reagent, synthesized as follows. (Caution! Chloroform, dichloromethane, dioxane, and hydrazine are carcinogenic in experimental animals! DMF may be carcinogenic! 3,5-Dinitrobenzoyl chloride and aluminum chloride are corrosive! Nitrobenzene is toxic!) 10 g Dried macroporous polystyrene (Xe-305, Rohm and Haas) + 10 g 3-nitro-4-chlorobenzyl alcohol + 1 O g anhydrous aluminum chloride + 50 mL nitrobenzene, heat at 65-70 for 3 days, cool, filter, wash polymer with three 50 mL portions of 1 M HCl in dioxane, with three 50 mL portions of DMF, with three 50 mL portions of MeOH, and with three 50 mL portions of dichloromethane, dry under vacuum at 100. Reflux 19 g of this polymer in 60 mL hydrazine hydrate:ethylene glycol monoethyl ether 40:60 for 20 h, cool to room temperature, filter off the polymer and wash it thoroughly with water. Suspend the polymer in 100 mL concentrated HChdioxane 50:50, reflux for 20 h, filter the polymer and wash it with five 100 mL portions of water, with three 100 mL portions of MeOH, and with three 50 mL portions of ether, dry under vacuum at 80. Functionalization was 1.17 mmoles/g (Eur.J.Biochem. 1975, 59, 55). Add a portion of polymer to dry chloroform, add a three-fold excess of 3,5-dinitrobenzoyl chloride and pyridine, stir at 0-10 for 30 min, filter off polymer, wash with chloroform to give the reagent (J.Org.Chem. 1984, 49, 922).)
HPLC VARIABLES

Guard column: 20 mm long Microsorb octadecyldimethylsilyl silica (Rainin) Column: 10 (sic)x 4.6 5 jxm Microsorb octadecyldimethylsilyl silica (Rainin) Mobile phase: MeCNrIO mM pH 2.5 phosphate buffer 45:55 Flow rate: 0.7 Injection volume: 10-20 Detector: UV 220
CHROMATOGRAM

Retention time: 16.3 Internal standard: l-methyl-3-phenylpropylamine (23.0) Limit of detection: 14 ng/mL Limit of quantitation: 47 ng/mL
OTHER SUBSTANCES

Noninterfering: benzoylecgonine, cocaine, codeine, glutethimide, imipramine, meperidine, methadone, methamphetamine, methaqualone, morphine, nortriptyline, oxazepam, phencyclidine, propoxyphene, quinine
KEYWORDS

SPE; derivatization
REFERENCE Fisher,D.H.; Bourque,A.J. Quantification of amphetamine in urine: solid-phase extraction, polymeric reagent derivatization and reversed-phase high-performance liquid chromatography, J. Chromatogr., 1993, 614, 142-147.

SAMPLE Matrix: urine

Sample preparation: 1 mL Urine + 0.5 mL 1% trichloroacetic acid, centrifuge at 5200 g for 10 min, filter (0.2 jjum), inject 20 fxL aliquot
HPLCVARIABLES

Column: 250 X 4 Lichrospher 5jjiin 60 RP-select B Mobile phase: Gradient. MeCN:50 mM pH 3.2 potassium phosphate buffer from 10:90 to 75:25 over 7 min, hold at 75:25 for 3 min, return to 10:90 over 5 min, equilibrate at 10: 90 for 5 min Flow rate: 1.5 Injection volume: 20 Detector: UV 190-370 CHROMATOGRAM Retention time: 4.9
OTHER SUBSTANCES

Extracted: amitriptyline, morphine, codeine, benzoylecgonine, meperidine, norpropoxyphene, nordiazepam Also analyzed: phenylpropanolamine, lidocaine, diphenhydramine, nortriptyline, ephedrine, cocaine (different gradient).
REFERENCE Li,S.; Gemperline,P.J.; Briley,K.; Kazmierczak,S. Identification and quantitation of drugs of abuse in urine using the generalized rank annihilation method of curve resolution, J.Chromatogr.B, 1994, 655, 213-223. SAMPLE

Matrix: urine Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 500 |xL MeOH and 500 |xL water. Adjust pH of urine to 10, centrifuge at 1500 g. 2 mL Supernatant + 100 |xL 75 jxg/mL p-phenylethylamine hydrochloride in water, add to the SPE cartridge, wash with 2.5 mL water, elute with 2 mL MeOH, evaporate the eluate to dryness. Reconstitute in water, add 500 |xL 8% sodium bicarbonate, add 500 JJLL 0.5% 1,2naphthoquinone-4-sulfonic acid sodium salt, make up to 1.5 mL with water, heat at 70 for 20 min, cool, add an equal volume of chloroform, shake for 2 min, centrifuge at 1500 g for 5 min. Remove the organic layer and dry it over anhydrous sodium sulfate, filter (0.45 |xm), inject a 25 |xL aliquot of the filtrate.
HPLC VARIABLES

Column: 125 X 4 5 |xm LiChrospher Si-60 Mobile phase: EtOH:chloroform:ethyl acetate:n-hexane 1:22:32:45 Flow rate: 2 Injection volume: 25 Detector: UV 280
CHROMATOGRAM

Retention time: 3.7 Internal standard: p-phenylethylamine hydrochloride (4.9)

OTHER SUBSTANCES

Extracted: methamphetamine
KEY WORDS

SPE; normal phase; derivatization

REFERENCE Campins Falcd,R; Molins Legua,C; Herraez Hernandez,R.; Sevillano Cabeza,A. Improved amphetamine and methamphetamine determination in urine by normal-phase high-performance liquid chromatography with sodium 1,2-naphthoquinone 4-sulphonate as derivatizing agent and solid-phase extraction for sample clean-up, J.Chromatogr.B, 1995, 663, 235-245. SAMPLE

Matrix: urine Sample preparation: Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH and 1 mL 50 mM pH 11 phosphate buffer. 500 |xL Urine + 500 |xL 2000 U/mL p-glucuronidase with sulfatase activity (TyPe H-I, Sigma) in 100 mM pH 5 acetate buffer, heat at 37 overnight, add 500 mg NaCl, add 500 |JLL 50 mM pH 11 potassium phosphate buffer, add 1.3 U | Lg 4'-hydroxymethamphetamine, add 3.1 |xg methamphetamine, adjust pH to 11 with ammonium hydroxide, mix. Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH and 1 mL 50 mM pH 11 phosphate buffer. Add the mixture to the SPE cartridge, wash with 1 mL 50 mM pH 11 potassium phosphate buffer, wash with 1 mL freshly prepared MeOH:water 30:70, wash with 1 mL MeCN, elute with 1 mL freshly prepared MeCN: acetic acid 98:2, elute with 1 mL MeCN:HCl 98:2. Combine the eluates and evaporate them to dryness under a stream of air at room temperature, reconstitute the residue in mobile phase (?), inject a 10 JULL aliquot.
HPLC VARIABLES

Guard column: phenyl Column: Microsorb phenyl Mobile phase: MeCN:MeOH:50 mM pH 3 potassium phosphate 5:10:85 Flow rate: 1 Injection volume: 10 Detector: UV 215
CHROMATOGRAM

Retention time: 12.2 Internal standard: 4'-hydroxymethamphetamine (6.0), methamphetamine (15.6) Limit of quantitation: 920 ng/mL
OTHER SUBSTANCES

Extracted: 4'-hydroxyamphetamine, metabolites

KEYWORDS

SPE; rat
REFERENCE Law,M.Y.L.; Moody,D.E. Simultaneous quantitation of amphetamine and 4'-hydroxyamphetamine by high performance liquid chromatography, J.Liq.Chromatogr., 1995, 18, 2029-2043. SAMPLE

Matrix: urine Sample preparation: Condition an Extra-Sep C18 SPE cartridge (Teknokroma) with 1 mL MeOH and 1 mL buffer. Adjust pH of 2 mL urine to ca. 10 with 100 u,L concentrated ammonium hydroxide, add 5 jig p-phenylethylamine, add to the SPE cartridge, wash with 5 mL water, wash with 1 mL MeCN, elute with 2 mL MeOH. Add 100 JJLL EtOHxoncentrated HCl 6:1 to the eluate, evaporate to dryness. Reconstitute with 1 mL buffer and 1 mL 0.5% l,2-naphthoquinone-4-sulfonic acid sodium salt, let stand at room temperature for 10 min, add 2 mL n-hexane:ethyl acetate 50:50, shake for 2 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 500 JLJLL MeCN:water 50:50, inject a 50 JJLL aliquot. (Buffer was 1% aqueous sodium bicarbonate adjusted to pH 10 with 5 M NaOH.)

HPLC VARIABLES

Column: 250 X 4 5 ^m Hypersil ODS-C18 Mobile phase: Gradient. MeCN:0.5% propylamine in water from 40:60 to 50:50 over 2.5 min, to 70:30 over 1 min, maintain at 70:30. Flow rate: 1 Injection volume: 50 Detector: UV 280
CHROMATOGRAM

Retention time: 4.7 Internal standard: p-phenylethylamine (4.1) Limit of detection: 4 ng/mL Limit of quantitation: 10 ng/mL OTHER SUBSTANCES Extracted: methamphetamine
KEYWORDS

SPE; derivatization
REFERENCE Molins Legua,C; Campins Falco,R; Sevillano Cabeza,A. Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with sodium 1,2-naphthoquinone 4-sulfonate as derivatizing agent and solid-phase extraction for sample clean-up, J.Chromatogr.B, 1995, 672, 81-88. SAMPLE

Matrix: urine Sample preparation: 100-300 \xL Urine + 100 |xL 1.5 M NaOH + 5 |xg IS, make up to 1 mL with water, add to an Extrelut 1 SPE cartridge, let stand for 20 min, elute with 6 mL benzene (Caution! Benzene is a carcinogen!). Add the eluate to 1 mL 100 mM sulfuric acid, extract. Remove the aqueous layer and add it to 3 mL 1.5 M NaOH, add 5 JJLL benzoyl chloride, vortex vigorously, extract twice with 3 mL portions of n-hexane. Combine the organic layers and wash them twice with 3 mL portions of water, evaporate the organic to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: Chiralcel OB-H Mobile phase: n-Hexane:isopropanol 90:10 Column temperature: 55 Flow rate: 1 Injection volume: 20 Detector: UV 220
CHROMATOGRAM

Retention time: 7.5 (L), 18 (D) Internal standard: 1-p-methoxyamphetamine (12) Limit of detection: 30 ng
OTHER SUBSTANCES

Extracted: ethylamphetamine, methamphetamine

KEYWORDS

rat; derivatization; SPE; chiral

REFERENCE Nagai,T.; Kamiyama,S.; Matsushima,K. Analysis of time-lapse changes of d- and 1-enantiomers of racemic ethylamphetamine and stereoselective metabolism in rat urine by HPLC determination, JAnal.ToxicoL, 1995, 19, 225-228. SAMPLE

Matrix: urine Sample preparation: 1 mL Urine + 10 mg p-glucuronidase/arylsulfatase (Helix pomatia, Sigma), heat at 37 overnight, add an equal volume of buffer, centrifuge at 2000 g for 5 min, inject an aliquot of the supernatant onto column A with mobile phase A and elute to waste. After 2.5 min backflush the contents of column A onto column B with mobile phase B, monitor the effluent from column B. Re-equilibrate both columns for 12.5 min before the next injection. (Buffer was 200 mM boric acid adjusted to pH 9.5 with 5 M NaOH.)
HPLC VARIABLES

Column: A 10 X 4.6 5 ^m Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120 (Shiseido) Mobile phase: A water; B Gradient. MeCN:buffer from 3:97 to 30:70 over 30 min, to 40:60 over 8 min (Buffer was 3.4 mL/L phosphoric acid adjusted to pH 3.0 with 5 M NaOH.) Flow rate: A 1.25; B 1 Injection volume: 100 Detector: UV 220
CHROMATOGRAM

Retention time: 9.2 Limit of detection: 250 ng/mL

OTHER SUBSTANCES

Extracted: acebutolol, alprenolol, atenolol, bopindolol, codeine, ephedrine, labetalol, metoprolol, morphine, nadolol, oxprenolol, pindolol, propranolol, timolol
KEYWORDS

column-switching
REFERENCE Saarinen,M.T.; Siren,H.; Riekkola,M.-L. Screening and determination of (3-blockers, narcotic analgesics and stimulants in urine by high-performance liquid chromatography with column switching, J.Chromatogr.B, 1995, 664, 341-346. SAMPLE

Matrix: urine Sample preparation: Dilute urine 20-fold or more. 500 |xL Diluted urine + 50 |xL 500 ng/ mL (+)-2,5-dimethoxyamphetamine hydrochloride + 100 |xL 500 mM NaOH + 2 mL benzene (Caution! Benzene is a carcinogen!), shake for 15 min, centrifuge at 1200 g for 5 min. Remove 1.8 mL of the organic phase and add it to 220 jxL 50 mM HCl, shake for 15 min, centrifuge at 1200 g for 5 min. Remove 200 (xL of the aqueous phase and add it to 40 jxL 250 mM NaOH, add 50 |xL 330 mM pH 7.8 phosphate buffer, add 250 uL MeCN, add 25 jxL 3 mM (-)-l-(9-fluorenyl)ethyl chloroformate, let stand at room temperature for 24 h, add 30 |ULL 100 mM glycine in water, let stand for 30 min, add 750 |xL pentane, vortex for 2 min, centrifuge at 1200 g for 5 min. Remove the organic layer and evaporate it to dryness in a centrifugal evaporator at room temperature, reconstitute the residue in 300 |xL MeCNrwater 50:50, inject a 100 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 3 juum Adsorbosphere HS C18 Mobile phase: MeCN:THF:20 mM pH 3.6 sodium acetate buffer 25:21:54

Flow rate: 1 Injection volume: 100 Detector: F ex 265 em 330

CHROMATOGRAM

Retention time: 37 (L), 40 (D) Internal standard: (+)-2,5-dimethoxyamphetamine (33) Limit of detection: 5 ng/mL OTHER SUBSTANCES Extracted: methamphetamine
KEY WORDS

rat; derivatization; chiral

REFERENCE Sukbuntherng,J.; Hutchaleelaha,A.; Chow,H.-H.; Mayersohn,M. Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC: Precolumn derivatization and fluorescence detection, JAnalToxicol, 1995, 19, 139-147. SAMPLE

Matrix: urine Sample preparation: Condition a 200 mg Bond Elut 18 SPE cartridge with 1 mL MeOH and 1 mL 1% pH 10 sodium bicarbonate buffer. 2 mL Urine + 400 |xL 8% pH 10 sodium bicarbonate buffer, mix, centrifuge at 1500 g for 2 min, add a 2 mL aliquot of the supernatant to the SPE cartridge, wash with 3 mL water, pass 500 |xL 2% sodium 1,2-naphthoquinone 4-sulfonate through the cartridge, pass 500 |xL 1% pH 10 sodium bicarbonate buffer through the cartridge, let stand at room temperature for 15 min, wash with 3 mL water, elute with 1 mL MeGNrwater 50:50, inject a 20 jxL aliquot of the eluate.
HPLC VARIABLES

Column: 250 X 4 5 xm Hypersil ODS Mobile phase: Gradient. MeCNrbuffer from 40:60 to 50:50 over 2.5 min, to 70:30 over 0.5 min, maintain at 70:30 for 1.5 min, to 85:15 over 1 min, maintain at 85:15 for 1.5 min. (Buffer was 5 mL/L propylamine in water.) Flow rate: 1 Injection volume: 20 Detector: UV 280
CHROMATOGRAM

Retention time: 4.1 Internal standard: p-phenylethylamine (3.6) Limit of detection: 100 ng/mL OTHER SUBSTANCES Extracted: methamphetamine
KEYWORDS

derivatization; SPE
REFERENCE Campins-Falc6,R; Sevillano-Cabeza,A.; Molins-Legua,C; Kohlmann,M. Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with simultaneous sample clean-up and derivatization with 1,2-naphthoquinone 4-sulphonate on solidphase cartridges, J.Chromatogr.B, 1996, 687, 239-246.

SAMPLE Matrix: urine

Sample preparation: Inject 15 |xL urine, inject a mixture of 5 jxL 20 mM 9-fluorenylmethyl chloroformate in MeCN and 45 |xL water, and inject 10 jxL buffer on to column A and elute to waste with mobile phase A. After 2.8 min backflush the contents of column A on to column B with mobile phase B and start the gradient, monitor the effluent from column B. At the end of the run condition column A with 1 mL mobile phase A. (Buffer was 4% sodium bicarbonate adjusted to pH 10 with 10% NaOH.)
HPLC VARIABLES

Column: A 20 X 2.1 30 fim Hypersil ODS-C18; B 125 X 4 5 jim LiChrospher 100 PR-C18 Mobile phase: A water; B Gradient. MeCN:water from 40:60 to 70:30 over 15 min. to 100: 0 over 5 min. Flow rate: A 0.35; B 1 Injection volume: 15 Detector: F ex 264 em 313
CHROMATOGRAM

Retention time: 15.3 Limit of detection: 10 ng/mL

OTHER SUBSTANCES

Extracted: ephedrine, methamphetamine, norephedrine, 3-phenylpropylamine, pseudoephedrine

KEYWORDS

column-switching; derivatization; on-column derivatization

REFERENCE Herraez-Hernandez,R.; Campins-Falc6,R; Sevillano-Cabeza,A. Determination of amphetamine and related compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chloroformate and liquid chromatography, J.Chromatogr.B, 1996, 679, 69-78. SAMPLE

Matrix: urine Sample preparation: Inject 50 |xL urine on to column A and elute to waste with mobile phase A, after 2 min inject a mixture of 25 fiL 0.5% sodium l,2-naphthoquinone-4-sulfonate in water and 25 (xL buffer on to column A, stop the flow of mobile phase A, after 10 min start pump A, after 5 min backflush the contents of column A on to column B with mobile phase B and start the gradient, monitor the effluent from column B. After each run flush column A with ethyl acetate for 1 min, n-hexane for 1 min, and ethyl acetate for 1 min, re-equilibrate with mobile phase A. (Buffer was 4% sodium bicarbonate adjusted to pH 10 with 10% NaOH.)
HPLC VARIABLES

Column: A 20 X 2.1 30 |xm Hypersil ODS-C18; B 250 X 4 5 jxm Hypersil ODS C18 Mobile phase: A water; B Gradient. MeCN:0.5% propylamine hydrochloride in water from 40:60 to 50:50 over 2.5 min, to 70:30 over 1 min, maintain at 70:30 for 4.5 min. Flow rate: 1 Injection volume: 50 Detector: UV 280
CHROMATOGRAM

Retention time: 5 Limit of detection: 25 ng/mL OTHER SUBSTANCES Extracted: methamphetamine

Next Page
KEY WORDS

column-switching; derivatization; on-column derivatization

REFERENCE Herrez-Hernridez,R.; Campins-Falc6,R; Sevillano-Cabeza,A. On-line derivatization into precolumns for the determination of drugs by liquid chromatography and column switching: Determination of amphetamines in urine, Anal.Chem., 1996, 68, 734-739. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 20 |xL 100 |JLM 1-phenylethylamine in water + 400 |xL concentrated HCl, heat at 80 for 1 h, cool, neutralize with 600 fxL 25% ammonia, add 5 mL 10% sodium carbonate solution, add 2 mL 500 mM pH 10.5 sodium borate buffer, add 2 mL chloroformrisopropanol 75:25, vortex for 1 min, centrifuge at 12.5 at 1500 g for 10 min, repeat the extraction. Combine the organic layers and remove a 100 |xL aliquot, add 10 |xL acetic acid to the aliquot, evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the residue in 50 |xL carbonate buffer, add 50 |xL 10 mM fluorescein-4-isothiocyanate in EtOH, mix, heat in the dark at 80 for 15 min, inject a 20 J U L L aliquot. (Prepare carbonate buffer by adjusting the pH of 200 mM sodium bicarbonate to 9.0 with 200 mM sodium carbonate.)
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Daisopak SP-120-5-ODS (Daiso, Osaka) Mobile phase: Gradient. MeCN:20 mM pH 7.9 sodium phosphate buffer 20:80 for 16 min then 24:76 (step-gradient). Flow rate: 0.8 Injection volume: 20 Detector: F ex 496 em 518
CHROMATOGRAM

Retention time: 32 Internal standard: 1-phenylethylamine (26.6) Limit of detection: 5.5 nM

OTHER SUBSTANCES

Extracted: metabolites, methamphetamine, norepinephrine

KEYWORDS

derivatization
REFERENCE Al-Dirbashi,O.; Kuroda,N.; Akiyama,S.; Nakashima,K. High-performance liquid chromatography of methamphetamine and its related compounds in human urine following derivatization with fluorescein isothiocyanate, J.Chromatogr.B, 1997, 695, 251-258.

Previous Page

Amphotericin
Molecular formula: C47H73NO17 Molecular weight: 924.09 CAS Registry No.: 1397-89-3 Merck Index: 627

SAMPLE

Matrix: CSF Sample preparation: Condition a BakerBond C18 SPE cartridge with 3 mL MeOH and 3 mL 100 mM pH 9 carbonate buffer. 1 mL CSF + 50 |xL 10 |xg/mL nystatin in MeOH, vortex briefly, add to the SPE cartridge, wash with 2 mL 100 mM pH 9 carbonate buffer, air dry for 2 min, elute with two 500 |xL aliquots of MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with 200 yJL MeOH, inject a 100 |xL aliquot.
HPLC VARIABLES

Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: MeCN:10 mM pH 5 EDTA 35:65 Flow rate: 0.5 Injection volume: 100 Detector: UV 410
CHROMATOGRAM

Retention time: 7.6 Internal standard: nystatin (8.5) Limit of detection: 0.5 ng/mL
KEY WORDS

dog; human; SPE; pharmacokinetics

REFERENCE Liu,H; Davoudi,H-; Last,T. Determination of Amphotericin B in cerebrospinal fluid by solid-phase extraction and liquid chromatography, J.Pharm.Biomed.AnaL, 1995, 13, 1395-1400. SAMPLE

Matrix: blood Sample preparation: Add 800 |xL cold MeOH to 200 (JLL serum, mix and centrifuge at 5 at 7000 rpm for 5 min. Filter (Millipore 0.45 |xm) the supernatant, inject a 5 jxL aliquot of the nitrate.
HPLC VARIABLES

Column: 33 X 4.6 1.5 um MICRANPS RP-C18 (Micra Sccientific, Northbrook, IL, USA) Mobile phase: MeCN:MeOH:50 mM sodium acetate 30:30:40 Flow rate: 0.7 Injection volume: 5 Detector: UV 382 CHROMATOGRAM Retention time: 2.7 Limit of detection: 0.2 ng Limit of quantitation: 0.625 ng

KEYWORDS

serum; dog; pharmacokinetics

REFERENCE Betto,R; Rajevic,M.; Bossu,E.; Gradoni,L. Improved assay for serum amphotericin-B by fast high performance liquid chromatography, J.Liq.Chromatogr.Rel.Technol., 1997, 20, 1857-1866. SAMPLE

Matrix: blood Sample preparation: Extract a 200-300 jxL aliquot of whole blood with DMSO:MeOH 2:1 or chloroform:MeCN:DMSO 30:30:40 containing IS. Mix for 30 s, allow the mixture to sit for 1 hour and repeat mixing. Centrifuge, filter (0.45 jjim) and inject a 50-100 JJIL aliquot of the supernatant. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES

Column: 150 X 3.9 10 jxm jjiBondapak C18 Mobile phase: MeCNiwater adjusted to pH 4.2 with EDTA 36:64 Flow rate: 1 Injection volume: 50-100 Detector: UV 405
CHROMATOGRAM

Internal standard: N-acetyl-amphotericin B Limit of quantitation: 75 ng/mL

KEYWORDS

pharmacokinetics; whole blood

REFERENCE Adedoyin,A.; Bernardo,J.E; Swenson,C.E.; Bolsack,L.E.; Horwith,G.; DeWit,S.; Kelly,E.; Klasterksy,J.; Sculier,J.R; DeValeriola,D.; Anaissie,E.; Lopez-Berestein,G.; Llanos-Cuentas,A.; Boyle,A.; Branch,R.A. Pharmacokinetic profile of ABELCET (amphotericin B lipid complex injection): combined experience from phase I and phase II studies, Antimicrob.Agents Chemother., 1997, 41, 2201-2208.

SAMPLE Matrix: blood Sample preparation: 200 JULL Serum + 600 JULL MeOH, vortex, centrifuge at 10500 g for 5 min, inject an 80 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 30 X 4.6 3 jim C18 (Perkin-Elmer) Mobile phase: MeCN:2.5 mM disodium EDTA 30:70 Flow rate: 1 Injection volume: 80 Detector: UV 405
CHROMATOGRAM

Retention time: 1.5 Limit of detection: 50 ng/mL

KEYWORDS

serum
REFERENCE Lopez-Galera,R.; Pou-Clave,L.; Pascual-Mostaza,C. Determination of amphotericin B in human serum by liquid chromatography, J.Chromatogr.B, 1995, 674, 298-300.

SAMPLE Matrix: blood

Sample preparation: Deproteinize serum with MeOH, inject an aliquot of the supernatant.
HPLCVARIABLES

Column: 30 mm long C18 Mobile phase: MeCN:2.5 mM disodium EDTA 30:70 Flow rate: 1 Detector: UV 405
CHROMATOGRAM

Retention time: 1.5 Limit of quantitation: 50 ng/mL

KEYWORDS

pharmacokinetics; serum
REFERENCE Ayestaran,A.; L6pez,R.M.; Montoro,J.B.; Estibalez,A; Pou,L.; Julia,A; Ldpez,A; Pascual,B. Pharmacokinetics of conventional formulation versus fat emulsion formulation of amphotericin B in a group of patients with neutropenia, Antimicrob.Agents Chemother., 1996, 40, 609-612. SAMPLE

Matrix: blood, fibrin clot Sample preparation: Digest fibrin clots with trypsin (1:1). 250 |xL Serum or digested fibrin clot + 250 |xL MeCN, vortex for 1 min, let stand at room temperature for 10 min, centrifuge at 2000 g, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 100 X 4.6 10 |xm Spherisorb C18-ODS2 Mobile phase: MeOH:5 mM EDTA 70:30 adjusted to pH 7.8 with 1M ammonium hydroxide Column temperature: 40 Flow rate: 1 Injection volume: 100 Detector: UV 385
CHROMATOGRAM

Retention time: 4.7 Limit of quantitation: 10 ng/mL

KEYWORDS

rabbit; serum; human; pharmacokinetics

REFERENCE Bouley,M.; Tod,M.; Chavanet,R; Petitjean,O. The penetration of amphotericin B from an Intralipid formulation into fibrin loci in a rabbit model of candidiasis, Biopharm.Drug Dispos., 1994, 15, 485-492. SAMPLE

Matrix: blood, tissue Sample preparation: Plasma. Mix 100 |xL plasma with 300 (xL MeOH, heat in a 50 water bath for 15 min, cool at room temperature for 5 min, centrifuge at 9500 g for 10 min. Inject a 50 |xL aliquot. Tissue. Mix 500 mg rat tissue with 4.5 mL MeOH and 500 \iL 10 mM pH 7.4 phosphate buffer. Homogenize for 5 min, vortex for 5 min, centrifuge until the supernatant is clear, inject a 120 |xL aliquot.

HPLC VARIABLES

Column: 150 X 4.6 5 |xm Hypersil ODS (plasma) or 216 X 4.6 10 |xm C18 (Whatman) (tissue) Mobile phase: MeOH :1 mM disodium EDTA containing 82 mM triethylamine and 96 mM phosphoric acid:deionized water 8:1:1.25 (plasma) or MeCN:10 mM sodium acetate 39.4: 60.6 Column temperature: 35.5 (tissue) Flow rate: 0.8 (plasma), 1 (tissue) Injection volume: 50 (plasma), 120 (tissue) Detector: UV 382
CHROMATOGRAM

Limit of quantitation: 50 ng/mL (plasma), 500 ng/g (tissue)

KEYWORDS

plasma; rat; pharmacokinetics; brain; kidney; liver; lung; spleen

REFERENCE Boswell,G.W.; BekerskyJ.; Buell,D.; Hiles,R.; Walsh,T.J. Toxicological profile and pharmacokinetics of a unilamellar liposomal vesicle formulation of amphotericin B in rats, Antimicrob.Agents Chemother., 1998, 42, 263-268. SAMPLE

Matrix: blood, tissue Sample preparation: Serum. 50-150 |xL Serum + 50 u,L 25 (xg/mL IS in MeOH + 2 mL MeOH:acetic acid 90:10, vortex for 30 s, leave in the dark for 1 h, centrifuge at 1000 g for 10 min, decant, filter (0.45 |xm), inject a 100 |xL aliquot. Tissue. 100-200 mg Tissue + 50 |xL 25 (xg/mL IS + 500 jxL 1 mM pH 7.4 phosphate buffer, vortex, homogenize using a manual glass homogenizer, add 2 mL MeOH:acetic acid 90:10, vortex for 30 s, leave in the dark for 1 h, centrifuge at 1000 g for 10 min, decant, filter (0.45 |xm), inject a 100 |xL aliquot.
HPLCVARIABLES

Guard column: 30 X 2 Alltech C18 Column: 300 X 3.9 10 |xm ixBondapak RP-C18 Mobile phase: MeCN: 10 mM pH 4.0 acetate buffer 37:63 Flow rate: 1 for 6 min, then 2 Injection volume: 100 Detector: UV 383
CHROMATOGRAM

Retention time: 15 Internal standard: natamycin (6) (UV 303) Limit of detection: 100 ng/mL Limit of quantitation: 1 |xg/mL
KEYWORDS

serum; lung; liver; mouse; pharmacokinetics

REFERENCE Polikandritou Lambros,M.; Abbas,S.A.; Bourne,D.W.A. New high-performance liquid chromatographic method for amphotericin B analysis using an internal method, J.Chromatogr.B, 1996, 685,135-140. SAMPLE

Matrix: blood, tissue Sample preparation: Homogenize 0.5 g tissue with 1 mL MeOH for 1 min. 100 [xL Serum or tissue homogenate + 100 JJLL cold MeCN, vortex for 10 s, centrifuge at 11800 g for 2 min, inject a 100 jxL aliquot of the supernatant.

HPLC VARIABLES

Column: 150 X 4.6 5 jxm Supelco LC-I Mobile phase: MeOH:5 mM EDTA buffer 65:35
CHROMATOGRAM

Limit of detection: 50 ng/mL

KEY WORDS

serum; rat; kidney; liver; lung

REFERENCE Wasan,K.M.; Vadiei,K.; Lopez-Berestein,G.; Luke,D.R. Pharmacokinetics, tissue distribution, and toxicity of free and liposomal amphotericin B in diabetic rats, J.Infect.Dis., 1990, 161, 562-566. SAMPLE

Matrix: blood, tissue Sample preparation: Homogenize 0.5 g tissue in 4.5 mL MeOH. Extract tissue homogenate or 0.5 mL plasma using a 1 mL Bond-Elut C18 SPE cartridge.
HPLC VARIABLES

Column: 300 X 3.9 10 juim fiBondapak C18 Mobile phase: MeCN:2.5 mM disodium EDTA 45:55 Detector: UV 382
CHROMATOGRAM

Limit of detection: =ss25 ng/g, =^5 ng/mL

KEYWORDS

plasma; rat; pharmacokinetics; liver; lung; kidney; spleen; heart; skin; lymph nodes; adrenal glands; thyroid; pancreas; testes; ileum; SPE
REFERENCE Fielding,R.M.; Smith,P.C; Wang,L.H.; Porter,J.; Guo,L.S. Comparative pharmacokinetics of amphotericin B after administration of a novel colloidal delivery system, ABCD, and a conventional formulation to rats, Antimicrob.Agents Chemother., 1991, 35, 1208-1213. SAMPLE

Matrix: blood, tissue, urine Sample preparation: Condition a 1 mL 100 mg Bond-Elut C18 SPE cartridge with 1-2 mL MeOH, with 1-2 mL water, and with 3 mL 10 mM pH 7.4 phosphate buffer. 0.5 mL Blood or 0.3-0.5 g tissue + 0.5 mL 10 mM pH 7.4 phosphate buffer, homogenize (Polytron homogenizer) for 5-10 s, add 4 mL MeOH, vortex for 30 s, centrifuge at 2000 g for 10 min. Add 0.5 mL plasma, 1-2 mL urine, or 0.5-2 mL supernatant from blood or tissue to 4 mL 10 mM pH 7.4 phosphate buffer, add this mixture to the SPE cartridge at < 1 mL/min, wash with 3 mL MeOH: 10 mM pH 7.4 phosphate buffer 40:60, centrifuge SPE cartridge at 2000 g for 2-3 min, elute with 0.75 (plasma) or 1 (others) mL MeCN:2.5 mM disodium EDTA 60:40 (plasma) or 50:50 (others), centrifuge to remove the last of the eluate, inject a 100 |xL aliquot of the eluate.
HPLC VARIABLES

Column: 300 X 3.9 10 |xm ixBondapak C18 Mobile phase: MeCN:2.5 mM disodium EDTA 45:55 Flow rate: 1 Injection volume: 100 Detector: UV 382

CHROMATOGRAM Retention time: 7

Limit of detection: =^25 ng/mL (blood), 2.5 ng/mL (urine), 50 ng/g (tissue), ^ 5 ng/mL (plasma)
KEYWORDS

plasma; SPE; rat; liver; kidney; lung; spleen; heart; brain; muscle; pharmacokinetics; stability
REFERENCE Wang,L.H.; Smith,P.C; Anderson,K.L.; Fielding,R.M. High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies, J.Chromatogr., 1992, 579, 259-268. SAMPLE

Matrix: blood, urine Sample preparation: Serum. 100 jxL Serum + 300 JULL 160 ng/mL IS in ice-cold MeOH, vortex, centrifuge. Remove the supernatant and concentrate it under a stream of nitrogen at room temperature, reconstitute the residue in 150 u,L MeOH, inject an aliquot. Urine. Dilute urine. 100 jxL Diluted urine + 300 |xL 160 ng/mL IS in ice-cold MeOH, vortex, inject an aliquot.
HPLCVARIABLES

Column: Nova-Pak C18 Mobile phase: MeCN:4 mM pH 7.0 Na2HPO4ZKH2PO4 buffer 31:69 Flow rate: 1.2 Detector: UV 405
CHROMATOGRAM

Retention time: 3.5 Internal standard: l-amino-4-nitronaphthalene (5.7) Limit of quantitation: 22 ng/mL
KEYWORDS

rat; serum; protect from light; pharmacokinetics

REFERENCE Chow,H.-H.; Wu,Y.; Mayersohn,M. Pharmacokinetics of amphotericin B in rats as a function of dose following constant-rate intravenous infusion, Biopharm.Drug Dispos., 1995, 16, 461-473. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min.

Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 346.2 CHROMATOGRAM Retention time: 15.718
KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: bronchoalveolar lavage fluid Sample preparation: Condition a Sep-Pak C18 SPE cartridge with two 3 mL portions of MeCN and two 3 mL portions of 10 mM pH 7.4 sodium acetate buffer. Mix the total volume of bronchoaspiration or bronchoalveolar lavage fluid with an equal volume of MeOH for 1 min, centrifuge at 3000 rpm for 20 min, combine the supernatant with an equal volume (?) of 10 mM pH 7.4 sodium acetate buffer, add to the SPE cartridge, wash five times with 3 mL portions of MeOHrIO mM pH 7.4 sodium acetate buffer 50:50, elute with two 1.5 mL portions of MeOH. Evaporate the eluate to dryness under nitrogen, reconstitute the residue in 400 jxL MeOH, vortex for 15 s, inject an aliquot.
HPLC VARIABLES

Column: 30 X 4.6 3 |xm Perkin-Elmer ODS Mobile phase: MeCN:2.5 mM EDTA disodium dihydrate 30:70 Flow rate: 1 Injection volume: 80 Detector: UV 405
CHROMATOGRAM

Retention time: 1.5 Limit of detection: 50 ng/mL

KEYWORDS

SPE
REFERENCE Lopez,R.; Pou,L.; Andres,L; Monforte,V.; Roman,A.; Pascual,C. Amphotericin B determination in respiratory secretions by reversed-phase liquid chromatography, J.Chromatogr.A, 1998, 812, 135-139. SAMPLE

Matrix: formulations Sample preparation: Dilute 1 mL samples to 5 or 10 mL with 5% dextrose, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 ^m Beckman ODS C18 Mobile phase: MeCN:MeOH:buffer 37:18:45 (Buffer was 50 mM sodium acetate and 3 mM disodium EDTA adjusted to pH 5.0 with glacial acetic acid.) Flow rate: 1.2 Injection volume: 20

Detector: UV 405
CHROMATOGRAM

Retention time: 7.8 Limit of detection: 625 ng/mL

OTHER SUBSTANCES

Simultaneous: amphotericin A, amphotericin X

KEYWORDS

injections; 5% dextrose; stability-indicating

REFERENCE Kintzel,P.E.; Kennedy,P.E. Stability of amphotericin B in 5% dextrose injection at concentrations used for administration through a central venous line, Am.J.Hosp.Pharm., 1991, 48, 283-285. SAMPLE

Matrix: formulations Sample preparation: Directly inject a 20 |xL aliquot.

HPLC VARIABLES

Column: 10 jjum Radial-Pak jxBondapak C18 Mobile phase: MeOH:MeCN:2.5 mM EDTA 50:35:20 Flow rate: 1.8 Injection volume: 20 Detector: UV 405
CHROMATOGRAM Retention time: 5.8 KEYWORDS

injections; 5% dextrose; stability-indicating

REFERENCE Mitrano,F.R; Outman,W.R.; Baptista,R.J.; Palombo,J.D. Chemical and visual stability of amphotericin B in 5% dextrose injection stored at 4 degrees C for 35 days, Am.J.Hosp.Pharm., 1991, 48, 26352637. SAMPLE

Matrix: formulations Sample preparation: 100 |xL Liposomal preparation + 100 jxL MeOH, vortex for 10 s, centrifuge at 13000 g for 2 min, inject a 75 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Supelco LC-I Mobile phase: MeOH:0.005% EDTA 65:35 Flow rate: 2 Injection volume: 75 Detector: UV 405
CHROMATOGRAM

Limit of detection: 50 ng/mL

KEYWORDS

liposomal preparations

REFERENCE Wasan,K.M.; Morton,R.E.; Rosenblum,M.G.; Lopez-Berestein,G. Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high-density lipoproteins: Role of lipid transfer protein, J.Pharm.ScL, 1994, 83, 1006-1010. SAMPLE

Matrix: solutions Sample preparation: Dissolve in DMSO to 10 mg/mL, dilute 1:20 with MeOH.
HPLC VARIABLES

Column: 250 X 4.6 10 |xm jxBondapak C18 Mobile phase: MeCN:50 mM phosphate buffer (pH 3.5-8.1) 30:70 to 35:65 Flow rate: 0.4-2 Detector: UV 313 OTHER SUBSTANCES Simultaneous: nystatin
KEY WORDS

for amphotericin A
REFERENCE Aszalos,A.; Bax,A.; Burlinson,N.; Roller,R; McNeal,C. Physico-chemical and microbiological comparison of nystatin, amphotericin A and amphotericin B, and structure of amphotericin A, J.Antibiot.(Tokyo), 1985, 38, 1699-1713. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH at a concentration of 15 mM, inject a 10 |JLL aliquot.
HPLC VARIABLES

Column: 50 X 4.6 3 |xm Econosphere C18 Mobile phase: MeOH:buffer 65:35 (Buffer was 6.3 g NaH2PO4 in 1 L water, adjust pH to 2.6 with phosphoric acid.) Column temperature: 50 Flow rate: 1 Injection volume: 10 Detector: UV 407 CHROMATOGRAM Retention time: 10
REFERENCE Backes,B.J.; Rychnovsky,S.D. A reverse-phase HPLC assay for measuring the interaction of polyene macrolide antifungal agents with sterols, Anal.Biochem., 1992, 205, 96-99. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: C18 Mobile phase: MeOHrDMF 20:80 Flow rate: 1.5 Injection volume: 20 Detector: UV 280

CHROMATOGRAM Retention time: 2.2 OTHER SUBSTANCES Simultaneous: cefpirome KEYWORDS

stability-indicating; protect from light

REFERENCE Allen,L.V.,Jr.; Stiles,M.L.; Prince,S.J.; Sylvestri,M.F. Stability of cefpirome sulfate in the presence of commonly used intensive care drugs during simulated Y-site injection, Am.J.Health-Syst.Pharm., 1995, 52, 2427-2433.

Ampicillin
Molecular formula: C16H19N3O4S Molecular weight: 349.41 CAS Registry No.: 69-53-4 (anhydrous), 32388-53-7 (monohydrate), 23277-71-6 (K salt), 7177-48-2 (trihydrate), 69-52-3 (Na salt) Merck Index: 628 LednicerNo.: 1 413, 2 437, 4 179

SAMPLE

Matrix: blood, tissue, urine Sample preparation: Plasma. 50 |xL Plasma + 50 |xL IS solution + 50 |xL MeCN, mix for 30 s, centrifuge at 5000 g for 15 min. Inject an aliquot of the supernatant. Urine. 100 |xL IS solution + 200 |JLL MeCN + 100 |xL urine, mix for 30 s, centrifuge at 5000 g for 15 min. Inject an aliquot. Tissue. Weight out finely chopped tissue and suspend it in 200 JJIL water. Add 100 |xL 100 |xg/mL IS, sonicate for 60 s. Add 200 |xL MeCN, vortex for 30 s, centrifuge at 10000 g for 15 min. Inject an aliquot.
HPLC VARIABLES

Guard column: 15 X 3.2 7 |xm Newguard C18 (Alltech) Column: 250 X 4.6 5 |xm Alltima C18 (Alltech) Mobile phase: MeCN:50 mM pH 5.0 sodium dihydrogen phosphate 10:90 Flow rate: 1.0 Detector: UV 215
CHROMATOGRAM

Retention time: 4.3 Internal standard: cefotaxime (11.6) Limit of quantitation: 500 ng/mL (plasma), 1 |xg/mL (urine), 2.5 |xg/g (tissue)
KEY WORDS

plasma; muscle; rat; pharmacokinetics

REFERENCE Cross,S.E.; Thompson,M.J.; Roberts,M.S. Distribution of systemically administered ampicillin, benzylpenicillin, and flucloxacillin in excisional wounds in diabetic and normal rats and effects of local topical vasodilator treatment, Antimicrob.Agents Chemother., 1996, 40, 1703-1710. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 JJLL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JXL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 \im Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min.

Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 3.827 KEYWORDS

whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: formulations Sample preparation: Capsules. Dissolve the powder from five capsules and the capsule shells in water, dilute to 1 L with water, filter an aliquot of the solution (0.2 jxm), dilute 10-fold with water, inject an aliquot. Syrup. Reconstitute the syrup powder, dilute 200fold with water, filter, inject an aliquot. Neonatal suspension. Reconstitute the powder, dilute 800-fold with water, filter, inject an aliquot.
HPLC VARIABLES

Column: 100 X 2.0 3 |xm Hypersil ODS Mobile phase: MeCN:20 mM phosphate buffer 15:85 containing 100 mM sodium dodecyl sulfate, adjusted to pH 2.0 with orthophosphoric acid Flow rate: 0.4 Injection volume: 20 Detector: UV 230
CHROMATOGRAM Retention time: 5.2 OTHER SUBSTANCES

Simultaneous: degradation products, cloxacillin

KEYWORDS

capsules; suspensions; syrup

REFERENCE Shakoor,O.; Taylor,R.B. Analysis of ampicillin, cloxacillin and their related substances in capsules, syrups and suspensions by high-performance liquid chromatography, Analyst, 1996, 121, 1473-1477. SAMPLE

Matrix: milk Sample preparation: Mix 10 mL milk with 2 mL 100 mM tetraethylammonium chloride, add 40 mL MeCN slowly with continual stirring, let stand for 10 min, decant the supernatant through a plug of glass wool. Collect 40 mL filtrate, add 2 mL buffer, evaporate to 1-2 mL under reduced pressure at 40-50, dilute to 4 mL with water, filter (0.45 jjim PVDF). Inject a 2 mL aliquot onto a 150 X 4.6 5 |xm Supelcosil LC-18 column, elute with MeCNrIO mM KH2PO4 0:100 for 3 min, to 60:40 over 37 min at 1 mL/min, collect a 1.52 mL aliquot containing the compound (ca. 18.5 min), evaporate to <1 mL under reduced pressure, add 200 |iL 10 mM KH2PO4 containing 10 mM phosphoric acid and 10 mM

sodium decanesulfonate, make up to 1 mL with water, inject an aliquot. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM Na2HPO4 in a 5:1 ratio, pH 6.)
HPLCVARIABLES

Column: 150 X 4.6 5 jxm Supelcosil LC-18 Mobile phase: MeCN:buffer 35:65 (Buffer was 10 mM phosphoric acid containing 5 mM potassium dihydrogen phosphate and 5 mM sodium dodecyl sulfate.) Flow rate: 1 Injection volume: 200 Detector: UV 215
REFERENCE Moats,W.A.; Romanowski,R-!D. Multiresidue determination of p-lactam antibiotics in milk and tissues with the aid of high-performance liquid chromatographic fractionation for clean up, J.Chromatogr.A, 1998, 812, 237-247. SAMPLE

Matrix: milk Sample preparation: Condition a 3 mL 500 mg Baker-10 C18 SPE cartridge (J.T. Baker) with 3 mL MeOH and 3 mL distilled water. Add 20 mL MeCN to 10 mL milk, vortex for 1 min, centrifuge at 1500 g for 10 min, concentrate the supernatant to 2-3 mL on a rotary evaporator at 40, add to the SPE cartridge, dry the cartridge under reduced pressure for 3 min, elute with 1 mL MeOH, filter (0.45 |xm) the eluate, inject a 10 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 ^m Kaseisorb LC ODS-300-5 (Tokyo Kasei) Mobile phase: MeCN:MeOH:50 mM KH2PO4 buffer 20:10:80 containing 5 mM sodium 1decanesulfonate, adjusted to pH 3.5 with concentrated phosphoric acid Column temperature: 40 Flow rate: 1 Injection volume: 10 Detector: UV 210
CHROMATOGRAM

Retention time: 7.5 Limit of detection: 30 ng/mL

OTHER SUBSTANCES

Extracted: cloxacillin, dicloxacillin, nafcillin, penicillin G

KEY WORDS

SPE
REFERENCE Takeba,K.; Fujinuma,K; Miyazaki,T.; Nakazawa,H. Simultaneous determination of p-lactam antibiotics in milk by ion-pair liquid chromatography, J.Chromatogr.At 1998, 812, 205-211. SAMPLE

Matrix: milk Sample preparation: Condition a 3 mL 500 mg Bond Elut C18 SPE cartridge with 20 mL MeOH, 20 mL water, and 10 mL 2% NaCl, do not allow to go dry. 5 mL Milk + 500 u.L 30% trichloroacetic acid, vortex thoroughly for 10 s, centrifuge at 0 at 3300 g for 30 min. Remove the liquid supernatant and add it to 100 |xL 4 M NaOH (to pH 5.2), vortex for 10 s, add 1 mL 20% NaCl, vortex for 10 s, add to the SPE cartridge at 3 mL/min, wash with 1 mL 2% NaCl, wash with 1 mL water, elute with 1 mL MeCN: 100 mM pH 6.5 phosphate buffer 40:60. Add 20 |xL 2 M NaOH to the eluate and vortex for 10 s (pH 8), add 10 |xL 200 mM acetic anhydride in MeCN, let stand for 3 min, add 500 |xL reagent,

vortex, heat at 65 for 10 min, cool to room temperature, inject a 200 |xL aliquot. (Prepare reagent by dissolving 13.78 g 1,2,4-triazole in 60 mL water, add 10 mL 100 mM mercuric chloride solution, mix, adjust pH to 9.0 0.5 with 4 M NaOH, make up to 100 mL with water.)
HPLC VARIABLES

Column: 150 X 3.9 4 fjun Nova-Pak C18 Mobile phase: MeCN:MeOH:buffer 18:12:70 (Prepare buffer by dissolving 4.969 g NaH2PO4, 10.139 g Na2HPO4.2H2O, 3.894 g sodium thiosulfate pentahydrate, and 6.791 g tetrabutylammonium hydrogen sulfate in 800 mL water, make up to 1 L with water.) Flow rate: 0.8 Injection volume: 200 Detector: UV 325
CHROMATOGRAM

Retention time: 16 Limit of detection: 3 ng/mL Limit of quantitation: 10 ng/mL

OTHER SUBSTANCES

Simultaneous: amoxicillin Noninterfering: cloxacillin, oxacillin, penicillin G

KEY WORDS

derivatization; SPE; cow

REFERENCE Verdon,E.; Couedor,P. Determination of ampicillin residues in milk by ion-pair reversed phase high performance liquid chromatography after precolumn derivatization, J.Pharm.Biomed.AnaL, 1996, 14, 1201-1207. SAMPLE

Matrix: milk Sample preparation: Condition a 500 mg tC18 SPE cartridge (Waters) with 20 mL MeOH, 20 mL water, and 10 mL 2% NaCl. Centrifuge 30 mL milk at 1500 g for 10 min. Dilute a 10 mL portion of the defatted milk with 20 mL water, add 200 |xL 2 jxg/mL penicillin V in pH 9.0 buffer, add 6 mL 170 mM sulfuric acid, add 5.6 mL 5% sodium tungstate, shake vigorously for 1 min, allow to stand for 5 min, check that the pH is in the range 4.6-4.8 (if it is outside this range start again using a different volume of sodium tungstate solution), centrifuge at 1500 g for 10 min, adjust the pH of the supernatant to 8.1-8.2 with 5 M and 0.1 M NaOH, filter (glass fiber) the clear liquid phase. Pass the filtrate through the SPE cartridge at 2 mL/min, wash with 2 mL water, dry by pulling air through the cartridge for 1 min, elute with 2 mL MeCN. Add 150 |xL pH 9.0 buffer to the eluate and evaporate to about 100 |xL under a stream of nitrogen at 45-50, add 400 (xL pH 9.0 buffer, add 75 (xL reagent I, vortex for 30 s, let stand at room temperature for 10 min, use 500 JJIL water to transfer the mixture to a separatory funnel, add 20 mL dichloromethane, add 5 mL pH 2.45 buffer, shake for 1 min, let stand for no more than 5 min. Remove the organic layer and evaporate it to dryness under reduced pressure at 35-40, dissolve the residue in 500 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex for 30 s, let stand at room temperature for 10 min, add 450 JULL reagent II, vortex for 1 min, heat at 55 1 for 30 min, cool, filter (0.45 |xm), inject a 150 fiL aliquot. (Prepare pH 9.0 buffer by dissolving 0.34 g KH2PO4 in water, adjusting the pH to 9.0 with NaOH, and making up to 100 mL with water. Prepare pH 2.45 buffer by dissolving 2.72 g KH2PO4 in water, adjusting the pH to 2.45 with phosphoric acid, and making up to 100 mL with water. Prepare reagent 1 by dissolving 1.13 g benzoic anhydride in MeCN, make up to 25 mL with MeCN. Prepare reagent II by dissolving 6.905 g 1,2,4-triazole in 30 mL water and adding 5 mL 26 mM mercuric chloride in water, adjust pH to 9.0 0.05 with 5 M NaOH,

make up to 50 mL. Prepare reagents I and II 1-4 h before use. Silanize glassware with dichlorodimethylsilane.)
HPLC VARIABLES

Column: 150 X 3.9 4 ^m Nova-Pak C18 Mobile phase: Gradient. A as MeCN:buffer 10:90. B was MeCN:buffer 30:70. A:B from 100: 0 to 0:100 over 30 min, maintain at 0:100 for 13 min, return to initial conditions over 2 min, re-equilibrate at initial conditions for 5 min. (Prepare buffer by dissolving 9.938 g Na2HPO4, 17.938 g NaH2PO4.H2O, and 4.964 g sodium thiosulfate in water, make up to 2 L with water, pH 6.5.) Column temperature: 30 Flow rate: 1 Injection volume: 150 Detector: UV 323
CHROMATOGRAM

Retention time: 32.5 Internal standard: penicillin V (28.5) Limit of detection: 1.5 ng/mL Limit of quantitation: 2.2 ng/mL
OTHER SUBSTANCES

Extracted: amoxicillin, cloxacillin, dicloxacillin, oxacillin, penicillin G

KEY WORDS

derivatization; cow; SPE

REFERENCE Sorensen,L.K.; Rasmussen,B.M.; Boison,J.O.; Keng,L. Simultaneous determination of six penicillins in cows' raw milk by a multiresidue high-performance liquid chromatographic method, J.Chromatogr.B, 1997, 694, 383-391. SAMPLE

Matrix: perfusate Sample preparation: Vortex perfusate, centrifuge at 11600 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES

Guard column: 20 X 2 5 jxm Hypersil ODS Column: 150 X 4.6 5 jim Hypersil ODS Mobile phase: MeOH:buffer 30:70 (Buffer was 50 mM KH2PO4 containing 0.1% triethylamine adjusted to pH 3 with orthophosphoric acid.) Flow rate: 1 Injection volume: 100 Detector: UV 229
CHROMATOGRAM

Retention time: 5.6 Limit of detection: 20 ng/mL Limit of quantitation: 100 ng/mL
REFERENCE Erah,P.O.; Barrett,D.A.; Shaw,P.N. Reversed-phase high-performance liquid chromatographic assay methods for the analysis of a range of penicillins in in vitro permeation studies, J.Chromatogr.B, 1998, 705, 63-69. SAMPLE

Matrix: solutions

HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere C18 Mobile phase: MeCNiI M acetic acid:l M KH2PO4:water 8:0.1:1:90.9 Flow rate: 1 Detector: UV 201
REFERENCE Walter,E.; Janich,S.; Roessler,B.J.; Hilfinger,J.M.; Amidon,G.L. HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: In vitro-in vivo correlation with permeability data from rats and humans, J.Pharm.Sci., 1996, 85, 1070-1076. SAMPLE

Matrix: tissue Sample preparation: Condition a 500 mg Isolute SCX SPE cartridge (Jones Chromatography, Hengoed, UK) with MeOH and water. Condition a 100 mg PGC (porous graphitic carbon) SPE cartridge (Shandon, Runcorn, UK) with acetone and pH 7.7 borate buffer. Add 20 mL water to 5 g tissue, homogenize, add 5 mL 170 mM sulfuric acid and 5 mL 5% aqueous sodium tungstate, mix well, centrifuge at 14000 g for 5 min. Discard pellet, add six drops orthophosphoric acid to the supernatant to adjust the pH to 2-2.5. Add it to the SCX SPE cartridge, allow to flow through the cartridge under vacuum at 2 mL/ min, wash with 5 mL 10 mM sulfuric acid, elute with 10 mL pH 7.7 borate buffer. Add the eluate to the PGC SPE cartridge. Wash with 5 mL water, place in-line filter (0.2 |xm, Anotop) below cartridge, elute with 20 mL acetone. Evaporate to dryness. Add 500 |xL water to the dry residue, add 20 JJLL 2% acetic anhydride in MeCN and let stand for 3 min. Add 500 |xL triazole/mercuric chloride derivatizing reagent and heat the mixture at 65 for 20 min. Inject a 100 |xL aliquot. (Borate buffer was 200 mM boric acid adjusted to pH 7.7 with 40% NaOH solution. Triazole/mercuric chloride reagent was prepared by mixing 34.45 g 1,2,4-triazole with 150 mL water and 25 mL 10 mM mercuric chloride, adjusted to pH 9.0 with 1 M NaOH and made up to 250 mL with water.) GL - 5 |xm Kromasil KR 100 C8 (Hichrom)
HPLCVARIABLES

Column: 250 X 3.2 5 p,m Kromasil KR 100 C8 (Hichrom) Mobile phase: MeCN:buffer 20:80 (Buffer was 15 mM potassium dihydrogen phosphate and 15 mM sodium thiosulfate) Flow rate: 0.55 Injection volume: 100 Detector: UV 325
CHROMATOGRAM

Retention time: 24.5 Limit of detection: 5 (xg/kg (muscle) Limit of quantitation: 50 jig/kg (muscle), 100 (xg/kg (liver)
OTHER SUBSTANCES

Extracted: amoxicillin
KEYWORDS

SPE; cow; liver; muscle; derivatization

REFERENCE Rose,M.D.; Tarbin,J.; Farrington,W.H.; Shearer,G. Determination of penicillins in animal tissues at trace residue concentrations: II. Determination of amoxicillin and ampicillin in liver and muscle using cation exchange and porous graphitic carbon solid phase extraction and high-performance liquid chromatography, Food Addit Con tarn., 1997, 14, 127-133.

SAMPLE Matrix: tissue

Sample preparation: Homogenize (Ultra-turrax T25) 5 g muscle with 14 mL 10 mM pH 4.5 sodium phosphate buffer at 10000 rpm for 2 min, add 1 mL 75% trichloroacetic acid in water, shake vigorously for 30 s, centrifuge at 3500 g for 10 min, filter (paper) the supernatant. Remove a 1 mL aliquot of the filtrate, add 200 |xL 20% trichloroacetic acid in water, add 200 jxL 7% formaldehyde in water, vortex for 20 s, heat at 100 for 30 min, cool to room temperature, make up to 2 mL with MeCN:water 20:80, inject a 100 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 [xm Prodigy ODS-3 (Phenomenex) Mobile phase: MeCN:20 mM pH 3.5 KH2PO4 buffer 25:75 Flow rate: 1 Injection volume: 100 Detector: F ex 346 em 422 CHROMATOGRAM Retention time: 12.5 Limit of detection: 0.6 ng/g Limit of quantitation: 1.5 ng/g
KEYWORDS

derivatization; muscle; cow; pig; chicken; fish; catfish

REFERENCE Luo,W.; Ang,C.Y.W.; Thompson,H.C.,Jr. Rapid method for the determination of ampicillin residues in animal muscle tissues by high-performance liquid chromatography with fluorescence detection, J.Chromatogr.B, 1997, 694, 401-407.

Amprolium
Molecular formula: C14H19CIN4 Molecular weight: 278.78 CAS Registry No.: 121 -25-5 Merck Index: 631 LednicerNo.: 1 264

SAMPLE

Matrix: blood Sample preparation: 200 |xL Plasma + 100 |xL 100 ng/mL IS in 330 mM perchloric acid + 500 |xL 330 mM perchloric acid, vortex for 30 s, centrifuge at 2150 g for 10 min. Remove the supernatant and allow it to stand for 3 h, inject a 30 jxL aliquot of the supernatant.
HPLC VARIABLES

Guard column: Sumipax PG-ODS-filter (Sumika, Osaka) Column: 250 X 4.6 5 |xm Capcell pack C18 UG-120 (Shiseido, Tokyo) Mobile phase: MeCN:200 mM KH2PO4 10:90 containing 5 mM sodium 1-hexanesulfonate Column temperature: 40 Flow rate: 0.6 Injection volume: 30 Detector: F ex 400 em 460 following post-column reaction. The column effluent mixed with the reagent pumped at 0.6 mL/min and the mixture flowed through a 10 m X 0.25 mm ID stainless steel coil at 40 to the detector. (Prepare reagent by dissolving 50 g NaOH and 800 mg potassium ferricyanide in 1 L water, store in the dark, discard after 24 h.)
CHROMATOGRAM

Retention time: 13 Internal standard: beclotiamine (3-[(4-amino-2-methyl-5-pyrimidinyl)methyl-5-(2-chloroethyl)-4-methylthiazolium chloride, Sankyo, Tokyo) (12) Limit of detection: 2 ng/mL Limit of quantitation: 5 ng/mL OTHER SUBSTANCES Extracted: metabolites, thiamine Noninterfering: ethopabate
KEYWORDS

post-column reaction; chicken; plasma; pharmacokinetics

REFERENCE HamamotOjK.; Koike,R.; Shirakura,A.; Sasaki,N.; Machida,Y. Rapid and sensitive determination of amprolium in chicken plasma by high-performance liquid chromatography with post-column reaction, J.Chromatogr.B, 1997, 693, 489-492. SAMPLE

Matrix: blood, tissue Sample preparation: Slurry 6 g alumina (alumina B Akt. I, ICN Biomedicals) in MeCN: MeOH 60:40, add to a 300 X 15 column, wash with 30 mL MeCN:MeOH 60:40. Homogenize (Niti-on Bio-mixer BM-2) 5 g chopped tissue or plasma with 25 mL MeCN for 2 min, wash twice with 20 mL portions of MeCN, filter (cotton plug), wash filter with 30 mL n-hexane saturated with MeCN, add 30 g anhydrous sodium sulfate to the filtrate, let stand at room temperature for 30 min, filter (cotton plug), add 30 mL isopropanol to the filtrate. Evaporate the filtrate to dryness at 35, reconstitute with 5 mL MeCN:MeOH 60:40, sonicate, add to the column, elute with 35 mL MeCN:MeOH 60:40. Add 10 mL

isopropanol to the eluate and evaporate it to dryness at 40, reconstitute with 1 jjig/mL chloramphenicol in mobile phase, filter (Gelman Ekikurodisk 13 CR), inject a 20 |xL aliquot of the nitrate.
HPLC VARIABLES

Column: 250 X 4.6 L-column ODS (Chemicals Inspection and Testing Institute, Tokyo) Mobile phase: MeCN:200 mM KH2PO4 15:85 containing 5 mM sodium 1-hexanesulfonate

Column temperature: 40 Flow rate: 0.7 Injection volume: 20

Detector: F ex 367 em 470 following post-column reaction. The column effluent mixed with the reagent pumped at 0.7 mL/min and the mixture flowed through a 10 m X 0.25 mm ID stainless steel coil at 40 to the detector. (Prepare reagent by dissolving 50 g NaOH in water, adding 800 mg potassium ferricyanide, and making up to 1 L with water.)
CHROMATOGRAM

Retention time: 8 Limit of detection: 2-4 ng/g

KEYWORDS

post-column reaction; chicken; muscle; liver; kidney; skin; plasma; SPE

REFERENCE Takahashi,Y.; Sekiya,T.; Nishikawa,M.; Endoh,Y.S. Simultaneous high-performance liquid chromatographic determination of amprolium, ethopabate, sulfaquinoxaline, and N4-acetylsulfaquinoxaline in chicken tissues, J.Liq.Chromatogr., 1994, 27, 4489-4512. SAMPLE

Matrix: eggs, tissue Sample preparation: Mix egg yolk with an equal amount of water, homogenize (Ultraturrax) for 30 s. Blend (Lameris Lab Blender 400 stomacher) 20-30 g tissue with twice the amount of water for 5 min, centrifuge at 460 g for 10 min. Dialyze (24" dialyzer with membrane Type C (Technicon, Tarrytown NY)) diluted egg yolk or tissue supernatant against water (both pumped at 0.6 mL/min), inject a 2 mL aliquot of the dialysate onto column A at 1 mL/min, wash with water at 1 mL/min for 6 min, backflush the contents of column A onto column B with mobile phase, after 2 min remove column A from the circuit, elute column B with mobile phase, monitor the effluent from column B.
HPLC VARIABLES

Column: A 50 X 4.6 37-50 |xm Corasil C18; B 20 mm long LC8 (Supelco) + 150 X 4.6 5 |xm Supelcosil LC8-DB Mobile phase: MeOH:water:acetic acidrtriethylamine 25:75:1:0.5 containing 5 mM heptanesulfonate

Column temperature: 40 Flow rate: 1 Injection volume: 2000

Detector: F ex 365 em 470 following post-column reaction. The column effluent mixed with the reagent pumped at 0.4 mL/min and the mixture flowed through a 3 m X 0.5 mm ID knitted PTFE coil to the detector. (Prepare reagent by dissolving 25 g NaOH and 160 mg potassium ferricyanide in 100 mL water.) CHROMATOGRAM Retention time: 10 Limit of detection: 3 ng/g
KEYWORDS

post-column reaction; column-switching; dialysis; yolk; muscle; chicken

REFERENCE van Leeuwen,W.; Wilhelmus van Gend,H. Determination of amprolium in egg yolk and muscle tissue (chicken) by HPLC with post-column reaction and fluorometric detection, using on-line sample cleanup and pre-concentration steps, Z.Lebensm. Unters.Forsch., 1988, 186, 500-504. SAMPLE

Matrix: feed, premix Sample preparation: Feed. 1 g Feed + 2 mL 5 |xg/mL thiamine monophosphate + 10 mL 5% sulfosalicylic acid + 10 mL hexane, vortex for 1 min, centrifuge at 2400 g for 10 min. Remove the aqueous layer and filter it (0.45 |xm, Gelman Aero LC 13). Inject a 360 |xL aliquot onto a 300 X 6 glass column packed with 200-400 mesh Dowex AG 2-X8 anion exchange resin, elute with 100 mM HCl at 1.2 mL/min, after about 2 min collect a 6-10 mL fraction, neutralize with 1 M NaOH (pH 7.0 0.5), inject a 500 JJLL aliquot of this fraction (J.Agric.Food Chem. 1980, 28, 1145). After 15-20 samples clean the sulfosalicylic acid from the column by backflushing with 700 mM NaCl containing 100 mM HCl .and 2% ferric chloride at 1.2 mL/min, flush for 20-30 min after the last of the iron chelate has gone (about 3 h). Re-equilibrate for 30 min. Premix. 0.8 mg Premix + 0.1 mg pyrithiamine + 10 mL water, grind (Omni-Mixer), add 10 mL hexane, grind for 5 min, centrifuge at 2400 g at 4 for 10 min. Filter the aqueous layer (0.45 jim, Millipore), dilute 20 times, inject an aliquot.
HPLC VARIABLES

Guard column: 30 X 4.6 5 |xm Rainin RP-18 guard column Column: 30 X 3 3 |xm Perkin-Elmer C18 Mobile phase: Feed. Gradient. 100 mM pH 5.5 sodium phosphate buffer for 6 min then 100 mM pH 2.6 sodium phosphate buffer for 19 min, re-equilibrate with original buffer for 15 min. Premix. Isocratic. 100 mM pH 2.6 Sodium phosphate buffer. Flow rate: 1 Injection volume: 10 Detector: F ex 339 em 432, following post-column reaction with 0.01% potassium ferricyanide in 15% NaOH pumped at 1 mL/min. The reagent is mixed with the column effluent and passed through a 7 m X 0.4 mm i.d. PTFE tube, kept at 32, to the detector.
CHROMATOGRAM

Retention time: 13.98 (feed), 4.03 (premix) Internal standard: thiamine monophosphate (5.26), pyrithiamine (1.62)
OTHER SUBSTANCES

Simultaneous: thiamine diphosphate, thiamine

REFERENCE Vanderslice,J.T.; Huang,M.-H.A. Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection, J.Assoc.Off.Anal.Chem., 1987, 70, 920-922. SAMPLE

Matrix: feed, premix Sample preparation: Weigh out 4-12 g feed, add 100 mL MeOH:water 2:1 containing 5 mM sodium dioctylsulfosuccinate and 10 mM calcium chloride, shake mechanically for 1 h, centrifuge, force 8-10 mL solution through a Sep-Pak alumina A SPE cartridge, discard first 3 mL effluent, collect next 4-6 mL, inject a 20 |xL aliquot.
HPLCVARIABLES

Column: 150 mm long 5 urn Econosphere C18 or 250 X 4.6 10 |xm Whatman ODS-3 Mobile phase: MeCN.water 40:60 containing 4 mM sodium dioctylsulfosuccinate, 0.3% diethylamine, and 1% acetic acid Flow rate: 1.5

Injection volume: 20 Detector: UV 270

CHROMATOGRAM

Retention time: 12 Limit of quantitation: 2500 ng/mL

OTHER SUBSTANCES

Noninterfering: ethopabate, sulfonamides, arsanilic acid, penicillin, streptomycin, chlortetracycline

KEYWORDS

rugged; SPE
REFERENCE Kentzer,E.J.; Cottingham,L.S.; Smallidge,R.L. Ion-pair reverse-phase liquid chromatographic determination of amprolium in complete feeds and premixes, J.Assoc.Off.Anal.Chem., 1988, 71, 251-255. SAMPLE

Matrix: tissue Sample preparation: Homogenize (Ultra-Turrax TP 18/10) 3 g tissue with 1 mL water and 4 mL acetone for 6 s, centrifuge at 5000 rpm for 3 min. Transfer 4 mL supernatant, add 5 mL dichloromethane, mix for 5 s, centrifuge at 3000 rpm for 3 min. Transfer the upper (water) layer to another tube and add 1 g NaCl, 3 mL MeCN, and 1 mL 300 mM NaOH. Shake vigorously for 20 s, centrifuge at 3000 rpm for 2 min, transfer the upper layer to another tube. Extract the remainder twice with 3 mL MeCN, discard the water layer. Evaporate the MeCN layers to dryness at 60 under a stream of nitrogen. Dissolve residue in 500 H L 20 mM KH2PO4 and filter (Spin-X) while centrifuging at 5600 g for 3 min.. Inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES

Guard column: 20 X 4.6 5 |xm Supelcosil LC-ABZ Column: 150 X 4.6 5 jjim Supelcosil LC-ABZ Mobile phase: MeCN:buffer 10:90 (Prepare buffer by dissolving 27.2 g KH2PO4 and 0.94 g hexane sulfonic acid sodium salt in 750 mL water, make up to 1 L with water.) Flow rate: 0.8 Injection volume: 20 Detector: F ex 365 em 470 following post-column reaction. The column effluent mixed in a vortex mixer with reagent pumped at 0.7 mL/min and this mixture flowed through a 10 m X 0.3 mm ID PTFE coil illuminated in a Beam Boost Photochemical Reactor to the detector. (The reagent was 1.25 M NaOH containing 25 mM potassium ferricyanide.)
CHROMATOGRAM

Retention time: 5.2 Limit of quantitation: 5 ng/g

KEYWORDS

chicken; muscle; post-column reaction; post-column photochemical derivatization

REFERENCE Hormazabal,V.; Yndestad,M. Rapid assay for the determination of residues of amprolium and ethopabate in chicken meat by HPLC, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 2517-2525.

SAMPLE Matrix: tissue Sample preparation: Prepare a cleanup column by plugging a 10 mm i.d. column with glass wool and adding 5 g activity I alumina, prewash with 70 mL MeCN:MeOH 90:10.

Homogenize 10 g minced chicken muscle with 50 mL MeOH at maximum speed (UltraTurrax T-18), filter through cotton, repeat extraction with another 50 mL MeOH. Combine filtrates, add 20 mL 1-propanol, concentrate to 3-4 mL under vacuum at 45. Add the residue to 20 mL MeCN and 50 mL n-hexane, shake vigorously by hand for 5 s, discard n-hexane layer, add another 50 mL n-hexane, shake for 5 min on a mechanical shaker, discard n-hexane layer. Evaporate the lower phase to dryness under vacuum at 45, dissolve the residue in 1 mL MeOH, add to cleanup column, wash with 30 mL MeCN:MeOH 90:10, elute with 30 mL MeCN:water 95:5. Evaporate the eluate to dryness under vacuum at 45, reconstitute the residue in 1 mL water, inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 7 u-m LiChrosorb RP-8 Mobile phase: MeCN:200 mM KH2PO4 20:80 containing 5 mM sodium 1-hexanesulfonate Column temperature: 30 Flow rate: 0.7 Injection volume: 10 Detector: F ex 367 em 470 following post-column reaction with 25 g NaOH and 0.4 g potassium ferricyanide in 500 mL water pumped at 0.7 mL/min. The column effluent and reagent were mixed and flowed through a 3 m X 0.3 mm i.d. stainless steel reaction coil to the detector.
CHROMATOGRAM

Retention time: 10 Limit of detection: 0.01 ppm

OTHER SUBSTANCES

Noninterfering: ethopabate, sulfonamides

KEYWORDS

chicken; muscle; SPE

REFERENCE Nagata,T.; Saeki,M. Liquid chromatographic determination of amprolium in chicken tissues, using postcolumn reaction and fluorometric detection, J.Assoc.Off.Anal.Chem.t 1986, 69, 941-943.

Amrinone
Molecular formula: C10H9N3O Molecular weight: 187.20 CAS Registry No.: 60719-84-8 Merck Index: 634 LednicerNo.: 3 147; 4 90, 115, 163

SAMPLE

Matrix: blood Sample preparation: Mix plasma or serum (minimum 50 jxL) with an equal volume MeCN, vortex for 15 s. Incubate at ambient temperature for 15 min, vortex for 15 s, centrifuge at 39 000 g for 2 min, inject a 25 jxL aliquot of the supernatant.
HPLC VARIABLES

Column: 100 X 2.1 5 |xm C18 narrow-bore (Hewlett-Packard) Mobile phase: Gradient. A was MeCN. B was 100 mm pH 6.0 phosphate buffer. A:B from 5:95 to 10:90 over 13.5 min. (Buffer was prepared by mixing 2 L 100 mm monobasic sodium phosphate with 280 mL 100 mM dibasic sodium phosphate.) Flow rate: 0.4 Injection volume: 25 Detector: UV 320, UV 345
CHROMATOGRAM

Retention time: 3.7 Limit of detection: 100 |Jig/L (320 nm), 500 jxg/L (345 nm)
OTHER SUBSTANCES

Extracted: metabolites Noninterfering: acetaminophen, bumetanide, carbamazepine, cefamandole, cefonicid, cefotaxime, cefoxitin, cefprozil, ceftazidime, ceftriaxone, ceftibuten, ceftizoxime, cefuroxime, cephalexin, cephalothin, desipramine, diazepam, digoxin, dobutamine, dopamine, doxepin, epinephrine, ethosuximide, fentanyl, furosemide, gentamicin, imipramine, methotrexate, midazolam, morphine, norepinephrine, phenobarbital, phenytoin, primidone, salicylic acid, theophylline, tobramycin, valproic acid, vancomycin (Monitoring at 345 nm may be necessary to avoid interference by cephalosporins.)
KEYWORDS

plasma; serum
REFERENCE Pappas,J.B.; Allen,E.M.; Ross,M.; Banner,W.,Jr. HPLC micromethod for amrinone and metabolites in patients receiving concurrent cephalosporin therapy, Clin.Chem., 1996, 42, 761-765. SAMPLE

Matrix: blood Sample preparation: Extract serum.

HPLC VARIABLES

Column: 150 X 3.3 7 jim Separon SGX Mobile phase: 80 mM ammonium perchlorate in MeOH Flow rate: 1 Detector: UV 254

CHROMATOGRAM Retention time: k' 0.89

OTHER SUBSTANCES

Simultaneous: nicotine, strychnine, neostigmine

KEYWORDS

serum
REFERENCE Eigendorf,H.G.; Nagel,S. Zur Analytik von Amrinone (Cordemcura). 2. Mitteillung: Hochdruckgliissigchromatographie [The analysis of amrinone (Cordemcura). 2. High pressure liquid chromatography], Pharmazie, 1987, 42, 631-631. SAMPLE

Matrix: blood Sample preparation: 100 fiL Plasma + 100 (xL ethyl acetate, vortex for 30 s, centrifuge at 2000 rpm for 5 min. Remove the organic phase and evaporate it under nitrogen at 30, reconstitute the residue in 100 JXL MeCN:5 mM pH 3.2 phosphate buffer 1:1, inject a 20 |xL aliquot.
HPLC VARIABLES

Guard column: Guard-pak cyano (Waters) Column: 100 X 8 4 ^m Nova Pak cyano Mobile phase: MeCN:5 mM pH 3.2 phosphate buffer 70:30 Flow rate: 2 Injection volume: 20 Detector: UV 210
CHROMATOGRAM

Retention time: 4.18 Limit of quantitation: 250 ng/mL

KEYWORDS

plasma
REFERENCE Bansal,R.; Louridas,A.T.; Gottesman,R.D.; Aranda,J.V. Determination of amrinone in human plasma by high-performance liquid chromatography with ultraviolet detection, J.Liq.Chromatogr., 1994, 17, 3531-3539. SAMPLE

Matrix: formulations Sample preparation: Dilute a 1 mL sample to 10 mL with mobile phase, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 10 |xm Partisil ODS III C18 Mobile phase: MeOH:water:0.5 M borate 40:58:2, pH 7.0 Flow rate: 2 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 3 OTHER SUBSTANCES Noninterfering: digoxin

KEYWORDS injections; stability-indicating; 5% dextrose; 0.45% NaCl REFERENCE Riley,C.M.; Junkin,P. Stability of amrinone and digoxin, procainamide hydrochloride, propranolol hydrochloride, sodium bicarbonate, potassium chloride, or verapamil hydrochloride in intravenous admixtures, Am.J.Hosp.Pharm., 1991, 48, 1245-1252.

Amsacrine
Molecular formula: C2IH19N3O3S Molecular weight: 393.47 CAS Registry No.: 51264-14-3 Merck Index: 635

SAMPLE

Matrix: blood Sample preparation: Add 100 JULL 20 JULM IS in MeOH to a glass tube and evaporate the MeOH to dryness under a stream of nitrogen at 35, add 500 JXL plasma, adjust pH to 3.0-4.0 with 120 |iL 500 mM HCl, vortex gently, add 5 mL hexane, shake for 20 min, centrifuge at 1720 g for 10 min. Remove the aqueous layer and adjust the pH to 9.0 with 500 |xL saturated sodium tetraborate, add 6 mL diethyl ether, shake for 15 min, centrifuge at 1720 g for 15 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 35, reconstitute the residue in 100 |xL MeOH, inject a 20-40 |xL aliquot.
HPLCVARIABLES

Column: 100 X 8 10 jjum Radial-Pak C18 (Waters) Mobile phase: MeCN:water:bufFer 396:594:10 (Buffer was 13.9 mL triethylamine in 60 mL water, adjust pH to 3.0 with phosphoric acid, dilute to 100 mL with water.) Flow rate: 7 Injection volume: 20-40 Detector: UV 254
CHROMATOGRAM

Retention time: 3.4 Internal standard: 4'-(3-methyl-9-acridinylamino)methanesulfonanilide (4.3) Limit of detection: 50 nM

OTHER SUBSTANCES

Noninterfering: doxorubicin, chlorambucil, cytosine arabinoside, 5-fluorouracil, lomustine, melphalan, methotrexate, prednisolone, 6-thioguanine, vincristine, vinblastine, degradation products
KEYWORDS

plasma; pharmacokinetics
REFERENCE Jurlina,J.L.; Paxton,J.W. High-performance liquid-chromatographic method for the determination of 4'(9-acridinylamino)methanesulfon-m-anisididein plasma, J.Chromatogr., 1983, 276, 367-374. SAMPLE

Matrix: blood, cells Sample preparation: 500 (xL Plasma, whole blood, or cells + 100 |xL 5 |xg/mL IS in 5% dextrose, adjust pH to 3.0-4.0 with 500 mM HCl, extract with hexane. Remove the aqueous layer and adjust the pH to 9.0 with saturated sodium tetraborate, extract with anhydrous diethyl ether. Remove the organic layer and evaporate it to dryness under a stream of air at 37, reconstitute the residue in 200 |xL MeOH: 148 mM phosphoric acid 50:10, centrifuge at 6000 g for 10 min, inject an aliquot.
HPLC VARIABLES

Guard column: 50 X 4.6 LiChrosorb RP-8 Column: 150 X 4.6 8 |xm Cp-Spher C8 (Chrompack)

Mobile phase: MeCN:water:buffer 396:594:10 (Buffer was 13.9 mL triethylamine in 60 mL water, adjust pH to 3.0 with 85% phosphoric acid, dilute to 100 mL with water.) Flow rate: 1.5 Injection volume: 100 Detector: UV 265
CHROMATOGRAM

Retention time: 2.8 Internal standard: N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino)phenylamino]-4acridinecarboxamide (CI-921) (3.5) Limit of detection: 6 ng/mL
KEY WORDS whole blood; plasma; pharmacokinetics REFERENCE Brons,RP.T.; Wessels,J.M.C; Linssen,P.C.M.; Haanen,C; Speth,P.A.J. Determination of amsacrine in human nucleated hematopoietic cells, J.Chromatogr., 1987, 422, 175-185. SAMPLE

Matrix: tissue Sample preparation: 1 g Tissue + 1 mL saline, mince, homogenize (Polytron PT-10) at 27000 rpm for 10-15 min, adjust pH to 2.0 with 500 mM HCl, centrifuge at 12000 g for 10 min. Add the supernatant to six volumes n-hexane, mix thoroughly. Remove the aqueous phase and adjust its pH to 9.0 with saturated sodium borate, extract with six volumes of ethyl acetate. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in MeOH:water 90:10, inject an aliquot.
HPLC VARIABLES

Column: 300 x 4 |jiBondapak C18 Mobile phase: MeOH:water:5% pH 4.3 sodium phosphate 450:50:3 Flow rate: 2 Detector: UV 254 CHROMATOGRAM Retention time: 10-12 Limit of detection: 200 ng/g
KEY WORDS

bladder; liver; lymph node; kidney; adrenal; ovary; stomach; thyroid; heart; lung; testicle; muscle; fat; spleen; pancreas; colon; prostate; brain; oncocytoma
REFERENCE Stewart,D.J.; Zhengang,G.; Lu,K; Savaraj,N.; Feun,L.G.; Luna,M.; Benjamin,R.S.; Keating,M.J.; Loo,T.L. Human tissue distribution of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (NSC 141549, AMSA), Cancer Chemother.PharmacoL, 1984, 12, 116-119.

Amylase
Molecular formula: indeterminate CAS Registry No.: 9000-92-4, 9000-85-5 (bacterial), 9000-90-2 (porcine), 9000-91-3 (sweet potato) Merck Index: 640 SAMPLE M a t r i x : blood

Sample preparation: Centrifuge at 100000 g for 15 min, inject a 200 (xL aliquot. Alternatively, filter (0.22 |xm cellulose nitrate), inject a 200 fxL aliquot.
HPLC VARIABLES

Guard column: 40 X 4 TSK SW Column: 300 X 8 GlasPac TSK 3000 SW Mobile phase: 10 mM pH 7.4 Phosphate buffer containing 135 mM NaCl Flow rate: 0.8 Injection volume: 200 (titanium injector) Detector: UV 280 or by enzyme activity
CHROMATOGRAM Retention time: 12-16 KEYWORDS

serum; GPC; SEC; human

REFERENCE Sion,J.-P.; Laureys,M.; Gerlo,E.; Gorus,F. Detection of macroenzymes in serum by high-performance gel permeation chromatography, J.Chromatogr., 1989, 496, 91-100.

Amylocaine
Molecular formula: C14H21NO2 Molecular weight: 235.33 CAS Registry No.: 532-59-2, 532-59-2 (HCI) Merck Index: 656

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 x 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben,

pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994, 18, 233-242.

Angiotensin Il
Molecular formula: C50H71N13O12 Molecular weight: 1046.19 CAS Registry No.: 4474-91-3 Merck Index: 689

SAMPLE M a t r i x : blood

Sample preparation: 10 |xL Serum + 240 JJLL 100 mM pH 7.5 potassium phosphate buffer containing 30 mM NaCl + 30 JJLL 1 M HCl, filter (0.2 ^m), inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Spherisorb ODS-C18 Mobile phase: MeOH:0.1% trifluoroacetic acid 60:40

Column temperature: 22 Flow rate: 1.5 Injection volume: 50 Detector: UV 220

CHROMATOGRAM

Retention time: 5.6 Limit of quantitation: 0.16 nmole

OTHER SUBSTANCES Extracted: angiotensin I KEYWORDS

serum; rat
REFERENCE Santos,R.A.S.; Krieger,E.M.; Greene,L.J. An improved fluorometric assay of rat serum and plasma converting enzyme, Hypertension, 1985, 7, 244-252. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL Analytichem weak cation-exchange (carboxymethylhydrogen form, CBA) SPE cartridge with 1 mL 1% trifluoroacetic acid in MeOH, 1 mL MeOH, and 2 mL water. Add 1 mL plasma to the SPE cartridge, rinse the tube with 1 mL water, add the rinse to the SPE cartridge, wash with 1 mL 1% trifluoroacetic acid in water, wash with 2 mL water, wash with 2 mL MeOH, elute with 2 mL 1% trifluoroacetic acid in MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeOH:buffer 50:50, inject a 5-75 |xL aliquot. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.) [Procedure was not necessarily validated for this compound.]
HPLC VARIABLES

Column: 250 X 2 5 |xm Ultrasphere octyl Mobile phase: Gradient. A was MeOH containing 10 mM sodium octanesulfonate. B was buffer containing 10 mM sodium octanesulfonate. A:B from 45:55 to 70:30 over 30 min,

maintain at 70:30 for 1 h. Alternatively isocratic at A:B 55:45 (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA in 1 L water, pH 3.2.) Column temperature: 60 Flow rate: 0.3 Injection volume: 5-75 Detector: F ex 390 em 470 following post-column reaction. The column effluent mixed with 400 mM NaOH pumped at 0.15 mL/min and 0.05% ninhydrin pumped at 0.05 mL/min and the mixture flowed through a 12 m X 0.33 mm i.d. reaction coil at 70 to the detector.
CHROMATOGRAM

Retention time: 21 (gradient), 66 (isocratic) Limit of detection: 100 fmole

OTHER SUBSTANCES

Simultaneous: adrenocorticotropin, angiotensin I, angiotensin III, atrial natriuretic peptide, bombesin, bradykinin, gonadorelin (LHRH), somatoliberin, vasopressin
KEYWORDS

plasma; SPE; post-column reaction

REFERENCE Rhodes,G.R.; Boppana,V.K. High-performance liquid chromatographic analysis of arginine-containing peptides in biological fluids by means of a selective post-column reaction with fluorescence detection, J.Chromatogr., 1988, 444, 123-131. SAMPLE

Matrix: blood Sample preparation: Condition a 100 mg Bond Elut phenyl SPE cartridge with 1 mL MeOH and 1 mL water. 6-8 mL Blood + 500 |iL inhibitor solution, cool in ice, centrifuge at 4, add 2 mL plasma to the SPE cartridge, wash with 1 mL water, elute with 500 (xL MeOH then 100 |JLL MeOH in to coated tubes, evaporate to dryness in a vacuum centrifuge evaporator, reconstitute with 140 |xL 100 mM acetic acid, centrifuge at 4 at 3000 g for 15 min, inject a 30-100 U | LL aliquot of the supernatant. (Inhibitor solution was EtOH.water 2:98 containing 25 mM phenanthroline, 125 mM disodium EDTA, 2 g/L neomycin, 1 mM enalaprilat, and 10 jxM pepstatin. Coat polypropylene tubes by filling with 5 g/L bovine serum albumin in 100 mM pH 7.5 Tris buffer containing 200 mg/L sodium azide overnight.)
HPLC VARIABLES

Guard column: 5 |xm Lichrospher RP-18 end-capped Column: 250 X 4.6 5 |xm Hypersil ODS Mobile phase: MeCN:86 mM pH 3.0 triethylammonium phosphate 21:79 (After 20 injections wash column with MeCN:water 80:20 for 100 min.) Column temperature: 45 Flow rate: 1 Injection volume: 30-100 Detector: UV 210 or immunoassay
CHROMATOGRAM R e t e n t i o n time: 6.4 OTHER SUBSTANCES

Simultaneous: angiotensin III

KEYWORDS

rat; SPE; plasma

REFERENCE Huang,H.; Baussant,T.; Reade,R.; Michel,J.B.; Corvol,P. Measurement of angiotensin II concentration in rat plasma: pathophysiological applications, Clin.Exp.Hypertens.fA]., 1989, 11, 1535-1548. SAMPLE

Matrix: blood Sample preparation: Add enalaprilat, 1,10-phenanthroline, and tripotassium EDTA to blood to give final concentrations of 3.6 jxM, 2.5 mM, and 1.5 mg/mL respectively, separate plasma by centrifuging at 4. 2 mL Plasma + 125angiotensin II, add to a hexane-washed, conditioned 500 mg Bond Elut C8 SPE cartridge, elute with two 1 mL portions of MeCN: water 50:50 containing 0.1% trifluoroacetic acid, filter the eluate, inject an aliquot of the filtrate.
HPLC VARIABLES

Column: 150 X 4.6 Dynamax C8 (Rainin) Mobile phase: Gradient. A was MeCN:water 20:80 containing 0.1% trifluoroacetic acid, adjusted to pH 4.0 with ammonium hydroxide. B was MeCN:water 35:65 containing 0.1% trifluoroacetic acid, adjusted to pH 4.0 with ammonium hydroxide. A:B from 100:0 to 0: 100 over 20 min. Column temperature: 45 Flow rate: 1 Detector: RIA
CHROMATOGRAM

Retention time: 11.9 Internal standard: 125angiotensin II (16)

OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III

KEYWORDS

plasma; SPE
REFERENCE Goldberg,M.R.; Tanaka,W.; Barchowsky,A.; Bradstreet,T.E.; McCrea,J.; Lo,M.-W.; McWilliams,E.J.,Jr.; Bjornsson,T.D. Effects of losartan on blood pressure, plasma renin activity, and angiotensin II in volunteers, Hypertension, 1993, 21, 704-713. SAMPLE

Matrix: enzyme incubations Sample preparation: 5 u,L Serum + 70 IULL 200 mM pH 7.5 phosphate buffer containing 30 mM NaCl + 20 |xL 800 |xM angiotensin I + 10 |iL water, heat at 37 for 15 min, add 100 |xL 500 mM perchloric acid, centrifuge at 800 g for 5 min. Remove a 100 uX aliquot of the supernatant and cool in ice-water, add 50 |xL 5 mM benzoin in 2-methoxyethanol, add 50 |JLL mercaptoethanol solution, add 100 |xL 0.8 M KOH, heat on a boiling water bath for 1.5 min, add 100 JJLL 1.2 M HC1:1 M pH 8.5 Tris-HCl buffer 50:50 (?) to adjust pH to 8.5, inject a 100 |mL aliquot. (Prepare mercaptoethanol solution by dissolving 780 mg (3-mercaptoethanol and 2.52 g sodium sulfite in 80 mL water, make up to 100 mL with water. The procedure measures the activity of angiotensin-converting enzyme in serum. The enzyme converts angiotensin I to angiotensin II which is then determined by HPLC.)
HPLC VARIABLES

Column: 250 x 4 5 jxm TSK gel ODS-120T (Toyo Soda) Mobile phase: MeOH:48 mM pH 8.5 phosphate buffer 33:67 Flow rate: 0.8 Injection volume: 100

Detector: F ex 325 em 435

CHROMATOGRAM

Retention time: 10 Limit of detection: 80-300 fmole OTHER SUBSTANCES Extracted: angiotensinl
KEYWORDS

derivatization
REFERENCE Sakamoto,Y.; Miyazaki,T.; Kai,M.; Ohkura,Y. Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fludrescence detection, J.Chromatogr., 1986, 380, 313-320. SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a 1 mg/g solution in water.

HPLC VARIABLES

Column: 250 X 4.6 5|xm Zorbax Rx C18 Mobile phase: MeCNrbuffer 20:80 (Buffer was 15 mM pH 7.0 phosphoric acid containing 5 mM hexadecyltrimethylammonium bromide.) Column temperature: 30 Flow rate: 1 Injection volume: 20 Detector: UV 220 CHROMATOGRAM Retention time: 4.5
OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III

REFERENCE Walker,T.A. Micellar HPLC: Investigation of the retention of positively charged peptides using cationic micellar mobile phases, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 1715-1727. SAMPLE

Matrix: solutions
HPLC VARIABLES Column: 300 X 3.9 10 |xm jxBondapak C18

Mobile phase: Gradient. A was 10 mM pH 4.15 ammonium acetate. B 1.5 mIVL acetic acid in MeOH. A:B from 80:20 to 25:75 over 45 min. Flow rate: 2 Detector: UV 210 CHROMATOGRAM Retention time: 23.0
OTHER SUBSTANCES

Simultaneous: angiotensin I, tetradecapeptide, tetrapeptide

REFERENCE Tonnaer,J.A.D.M.; Verhoef,J.; Wiegant,V.M.; de Jong,W. Separation and quantification of angiotensins and some related peptides by high-performance liquid chromatography, J.Chromatogr., 1980, 183, 303-309. SAMPLE

Matrix: solutions Sample preparation: Inject a 1-10 u,L aliquot of an aqueous solution.
HPLC VARIABLES

Guard column: 30 X 4.6 Micro-Guard ODS-10 (Bio-Rad) Column: 150 x 4 10 |xm Bio-Sil ODS-10 (Bio Rad) Mobile phase: MeCN:50 mM NaH2PO4 25:75, pH 6.0 Flow rate: 1 Injection volume: 1-10 Detector: UV 210
CHROMATOGRAM Retention time: 3.3 OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III

REFERENCE Guy,M.N.; Roberson,G.M.; Barnes,L.D. Analysis of angiotensins I, II, III, and iodinated derivatives by high-performance liquid chromatography, Anal.Biochem., 1981, 112, 272-277. SAMPLE

Matrix: solutions Sample preparation: Cool in ice while mixing 100 jxL of an aqueous solution, 50 fxL 5 mM benzoin in 2-methoxyethanol, 50 |xL mercaptoethanol solution, and 100 (xL 0.8 M KOH, heat on a boiling water bath for 1.5 min, add 100 JJLL 1.6 M HC1:1 M pH 8.5 Tris-HCl buffer 50:50, inject a 100 jxL aliquot. (Prepare mercaptoethanol solution by dissolving 780 mg p-mercaptoethanol and 2.52 g sodium sulfite in 80 mL water, make up to 100 mL with water.)
HPLCVARIABLES

Column: 15 X 4 (sic) 5 ^m LiChrosorb RP-18 Mobile phase: MeCN:50 mM pH 8.5 phosphate buffer 31:69 Flow rate: 0.8 Injection volume: 100 Detector: F ex 325 em 425
CHROMATOGRAM

Retention time: 5 Limit of detection: 27 fmole

OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III, gonadorelin, leupeptin acid, substance P, tuftsin

KEYWORDS

derivatization
REFERENCE Kai,M.; Miyazaki,T.; Sakamoto,Y.; Ohkura,Y. Use of benzoin as pre-column fluorescence derivatization reagent for the high-performance liquid chromatography of angiotensins, J.Chromatogr., 1985, 322, 473-477.

SAMPLE

Matrix: solutions Sample preparation: 200 |xL Solution + 100 |xL chloroform + 50 fiL 3 M KOH, heat at 60 for 10 min, cool in ice-water for 1 min, add 50 |xL 14 M acetic acid, add 300 fiL freshly prepared 265 jig/mL l,2-diamino-4,5-dimethoxybenzene monohydrochloride in water (with cooling in ice-water), heat at 60 for 18 min, cool, inject a 100 |xL aliquot. (Prepare l,2-diamino-4,5-dimethoxybenzene monohydrochloride as follows. Stir 483 g veratrole in 1.45 L acetic acid at 15 for 1 h, add 683 g concentrated nitric acid (d 1.05) over 1 h (maintain the temperature below 40 by cooling and regulating the rate of addition of the nitric acid). Continue stirring and add 2.127 L fuming nitric acid (d 1.50) over 1 h while maintaining the temperature below 30, let stand for 2 h, pour into a large volume of cold water, filter, wash the solid with water until the washings are neutral, recrystallize from EtOH to give 4,5-dinitroveratrole (mp 129.5-130.5) (J. Am. Chem. Soc. 1946, 68, 1536). Reflux 5 g 4,5-dinitroveratrole in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh iron powder and 20 mL concentrated HCl in small portions over J. h, reflux for 4 h, add 10 mL water, reflux for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with 200 mL portions of benzene. Combine the organic layers and evaporate them to dryness, add 10 mL concentrated HCl, recrystallize from EtOH to give l,2-diamino-4,5-dimethoxybenzene monohydrochloride as very slightly pink needles (mp 240) (Anal. Chim. Acta 1982, 134, 39).)
HPLC VARIABLES

Column: 150 X 4 5 |xm LiChrosorb RP-18 Mobile phase: MeCN:buffer:50 mM sodium 1-hexanesulfonate 26:64:10 (Prepare buffer by dissolving 14.9 g KCl in 950 mL water, adjusting pH to 2.2 with concentrated HCl, and making up to 1 L with water.) Flow rate: 0.8 Injection volume: 100 Detector: F ex 350 em 425
CHROMATOGRAM

Retention time: 14.5 Limit of detection: 11.3 pmole

OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III, leucine enkephalin, methionine enkephalin

KEY WORDS

derivatization; specific for tyrosine-containing peptides

REFERENCE Ishida,J.; Kai,M.; Ohkura,Y. High-performance liquid chromatography of tyrosine-containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene, J.Chromatogr., 1986, 356, 171-177. SAMPLE

Matrix: solutions Sample preparation: Inject a 10-50 uX aliquot.

HPLC VARIABLES

Column: 200 X 4 5 |xm TSKgel ODS-120T (Toyo Soda) Mobile phase: Gradient. A was MeCN:200 mM pH 2.3 sodium phosphate buffer 5:95. B was MeCN:200 mM pH 2.3 sodium phosphate buffer 40:60. A:B 100:0 for 5 min, to 68.6: 31.4 step gradient), to 35.7:64.3 over 45 min. Flow rate: 1 Injection volume: 10-50 Detector: F ex 325 em 435 following post-column reaction. The column effluent mixed with reagent A pumped at 1 mL/min and the mixture flowed through a 15 m X 0.3 mm ID

PTFE coil at 76. The effluent from this coil mixed with reagent B pumped at 0.4 mL/ min and this mixture passed through a 10 X 4 column packed with 40 mg glass wool. (Prepare reagent A by mixing equal volumes of 6 mM benzoin in 2-methoxyethanol, 4.8 M KOH, and 2.1 M p-mercaptoethanol. Prepare reagent B by mixing equal volumes of 1 M Tris and 4.2 M HCl.)
CHROMATOGRAM

Retention time: 20 Limit of detection: 5-15 pmole

OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin III, kallidin, kyotorphin, p-melanocyte stimulating hormone, substance P Noninterfering: estriol, estrone-3-sulfate, methionine enkephalin, phenylalanine, phenylpyruvic acid, propionic acid, sorbic acid, tryptophan, tyrosine
KEYWORDS

post-column reaction
REFERENCE Ohno,M.; Kai,M.; Ohkura,Y. On-line post-column fluorescence derivatization of arginine-containing peptides in high-performance liquid chromatography, J.Chromatogr., 1987, 392, 309-316. SAMPLE

Matrix: solutions Sample preparation: Dry a 5 |xL aliquot of a 0.1-4 mM aqueous solution, reconstitute with 100 |xL pH 9.5 100 mM lithium carbonate/sodium bicarbonate buffer, add 100 (JLL 15 mM 9-fluorenylmethyl chloroformate in acetone, let stand at room temperature for 30 s, wash with five 500 |xL portions of n-pentane. Remove a 10 u.L aliquot of the aqueous phase and add it to 90 jxL 10 mM NaH2PO4, mix, inject a 2 JJLL aliquot.
HPLC VARIABLES

Column: 125 X 2 5 |xm LiChrospher RP-2 Mobile phase: MeCNrIO mM pH 4.9 NaH2PO4 54:46 Flow rate: 1 Injection volume: 2 Detector: F ex 260 em 310
CHROMATOGRAM

Retention time: 5.2 Limit of detection: 500 fmole

KEYWORDS

derivatization
REFERENCE Vogt,W.; Egeler,E.; Sommer,W.; Eisenbeiss,F.; Meyer,H.D. High-performance liquid chromatographic determination of hormonal peptides and their fluorenylmethoxycarbonyl derivatives, J.Chromatogr., 1987, 400, 83-89. SAMPLE

Matrix: solutions Sample preparation: Mix a 20 (xL aliquot with 20 jxL 500 mM pH 9.0 potassium phosphate buffer and 30 |xL 2 mg/mL fluorescamine, mix, let stand at room temperature for 5 min, inject a 10-50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 2 5 |jim Ultrasphere octylsilica

Mobile phase: Gradient. MeOH:buffer from 40:60 to 65:35 over 10 min, maintain at 65:35 for 10 min. (Prepare buffer by dissolving 1.21 g Tris and 2.8 mL triethylamine in 1 L water, adjust pH to 7.0 with phosphoric acid.) Column temperature: 50 Flow rate: 0.3 Injection volume: 10-50 Detector: F ex 390 em 470 (cutoff filter) CHROMATOGRAM Retention time: 10
OTHERSUBSTANCES

Simultaneous: angiotensin I, angiotensin III

KEYWORDS

derivatization
REFERENCE Boppana,V.K.; Miller-Stein,C; Politowski,J.E; Rhodes,G.R. High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine, J.Chromatogr., 1991, 548, 319-327. SAMPLE

Matrix: solutions Sample preparation: Mix 20 |JLL of a 25 |xM peptide solution in water with 50 |ULL 200 mM ascorbic acid in water, 100 |xL 10 mM NaCN in water, 200 |JLL 5 mM naphthalene-2,3dicarboxaldehyde in MeCN, and 540 jxL 100 mM pH 7.0 phosphate buffer, mix, let stand at 0-4 for 20 min, add 50 |JLL 200 mM taurine in water, inject an aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm ODS Hypersil Mobile phase: THF:100 mM pH 6.0 phosphate buffer 19:81 containing 30 mM sodium 1octanesulfonate Column temperature: 40 0.1 Flow rate: 1-2 Detector: F ex 420 em 490
CHROMATOGRAM

Retention time: k' 11 Limit of detection: 50-100 fmole OTHER SUBSTANCES Simultaneous: similar peptides
KEYWORDS

derivatization
REFERENCE Patel,H.B.; Stobaugh,J.R; Riley,C.M. Reversed-phase ion-pair liquid chromatography of the angiotensins, J.Chromatogr., 1991, 536, 357-370.

SAMPLE Matrix: solutions

HPLC VARIABLES

Column: 250 X 4.5 5 |xm Kromasil C8 (Eka-Nobel)

Mobile phase: Gradient. A was MeCN:water 10:90 containing 0.1% trifluoroacetic acid. B was MeCN:water 90:10 containing 0.1% trifluoroacetic acid. A:B from 0:100 to 75:25 over 8 min, to 25:75 over 12 min. Flow rate: 2 Detector: UV 254 CHROMATOGRAM Retention time: 8.7
OTHER SUBSTANCES

Simultaneous: angiotensin I, bradykinin, insulin, leucin enkephalin, lysozyme, melittin, methionine enkephalin, oxytocin
REFERENCE Supelco Catalog, 1992, p. 104.

SAMPLE Matrix: solutions

HPLC VARIABLES

Column: 75 X 4.6 5 jxm Hypersil WP 300-octyl Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic acid in n-propanol. A:B 100:0 for 2.5 min, to 90:10 over 2.5 min, to 10:90 over 112 min. Flow rate: 1 Detector: UV 225 CHROMATOGRAM Retention time: 15
OTHER SUBSTANCES

Simultaneous: bombesin, bovine growth hormone, bovine serum albumin, catalase, lysozyme, L-tryptophan
REFERENCE Supelco Catalog, 1993, p. 602.

SAMPLE Matrix: solutions

HPLC VARIABLES

Column: 250 X 4.6 TSKgel ODS-120T Mobile phase: Gradient. A was MeOH:water 20:80 containing 0.05% trifluoroacetic acid. B was MeOH.water 50:50 containing 0.05% trifluoroacetic acid. A.B from 100:0 to 0:100 over 1 h. Flow rate: 1 Detector: UV 220 CHROMATOGRAM Retention time: 12
OTHERSUBSTANCES

Simultaneous: angiotensin I, calcitonin (human), a-endorphin, p-endorphin, gonadorelin (LH-RH), protirelin (TRH), somatostatin
REFERENCE Varian Catalog, 1993, p. 182.

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 10 \xm Vydac 228TP Mobile phase: Gradient. A was 0.25% trifluoroacetic acid in water. B was 0.25% trifluoroacetic acid in MeCN:water 70:30. A:B from 95:5 to 0:100 over 30 min. Flow rate: 1.5 Detector: UV 220 CHROMATOGRAM Retention time: 11
OTHER SUBSTANCES

Simultaneous: angiotensin I, bradykinin, eledosin, insulin, lysozyme, myoglobin, neurotensin, ovalbumin, oxytocin
REFERENCE Supelco Catalog, 1993, p. 581. SAMPLE

Matrix: solutions Sample preparation: Prepare a 1 mM solution in 100 mM pH 6 morpholinoethanesulfonic acid/NaOH buffer. Remove a 20 jxL aliquot and add it to 5 |xL 25 mM 5-carboxytetramethylrhodamine succinimidyl ester (Molecular Probes, Eugene OR) in DMF, let stand at room temperature overnight, add 50 JJLL 1 M pH 8 Tris-HCl buffer, inject a 5 |xL aliquot.
HPLCVARIABLES

Guard column: reversed-phase Column: reversed-phase Mobile phase: Gradient. A was MeCN containing 0.1% trifluoroacetic acid. B was water containing 0.1% trifluoroacetic acid. A:B from 5:95 to 55:45 "applied just after application of a sample". Flow rate: 1 Injection volume: 5 Detector: UV 280 CHROMATOGRAM Retention time: 38 OTHER SUBSTANCES Simultaneous: angiotensin I
KEYWORDS

derivatization
REFERENCE Shimura,K; Kasai,K.-I. Fluorescence-labeled peptides as isoelectric point (p/) markers in capillary isoelectric focusing with fluorescence detection, Electrophoresis, 1995, 16, 1479-1484. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 5 |mm Zorbax 300 A SB-C3

Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 5:95:0.1. B was MeCN: water:trifluoroacetic acid 5:95:0.085. A:B from 85:15 to 47:53 over 20 min. Column temperature: 35 Flow rate: 1 Injection volume: 10 Detector: UV 215
CHROMATOGRAM Retention time: 5 OTHER SUBSTANCES

Simultaneous: carbonic anhydrase, cytochrome C, insulin, leucine enkephalin, lysozyme, myoglobin, RNAase
REFERENCE Ricker,R.D.; Sandoval,L.A.; Permar,B.J.; Boyes,B.E. Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis, J.Pharm.BiomedAnal., 1996, 14, 93-105. SAMPLE

Matrix: tissue Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH, 10 mL THF, 10 mL hexane, 10 mL MeOH, and 10 mL water. Homogenize (Polytron) tissue with 20 volumes of ice-cold EtOH: 180 mM HCl 75:25, centrifuge at 4 at 32570 g for 20 min, store the supernatant at -20 for 18-20 h, centrifuge at 4 at 2200 g for 20 min. Adjust the pH of the supernatant to 6.5 with 1 M NaOH, store at 4 for 1 h, centrifuge at 4 at 2200 g for 20 min, evaporate to dryness under reduced pressure, reconstitute with 10 mL pH 3 0.1% trifluoroacetic acid, add to the SPE cartridge, wash with water, wash with 10 mL MeCN:0.1% trifluoroacetic acid 10:90, elute with 10 mL MeCN:0.1% trifluoroacetic acid 30:70, inject a 50-200 jxL aliquot of the eluate.
HPLC VARIABLES

Column: 150 X 3.9 4 |xm Nova Pak C18 Mobile phase: Gradient. A was MeCN:25 mM pH 7.6 phosphate buffer 5:95. B was MeCN: 25 mM pH 7.6 phosphate buffer 95:5. A:B from 89:11 to 68:32 over 12 min (Waters curve 7). Flow rate: 1.5 Injection volume: 50-200 Detector: UV 214 or radioimmunoassay
CHROMATOGRAM Retention time: 8 KEYWORDS

rat; adrenal; brain; heart; kidney; liver; lung; ovary; plasma; testes; uterus; SPE
REFERENCE De Silva,P.E.; Husain,A.; Smeby,R.R.; Khairallah,P.A. Measurement of immunoreactive angiotensin peptides in rat tissues: some pitfalls in angiotensin II analysis, Anal.Biochem., 1988, 174, 80-87.

Aniracetam
Molecular formula: C12H13NO3 Molecular weight: 219.24 CAS Registry No.: 72432-10-1 Merck Index: 700

SAMPLE Matrix: blood Sample preparation: 50 |xL Plasma, 100 |xL 50 |x g/mL IS in MeCN, mix, centrifuge at 12000 rpm for 10 min. Filter (0.45 |JL m) the supernatant, inject an aliquot of the filtrate.
HPLCVARIABLES

Column: 150 X 4.6 Inertsil ODS Mobile phase: MeCN:water:acetic acid 25:75:0.5 Flow rate: 1.0 Detector: UV 254 CHROMATOGRAM Internal standard: aspirin Limit of detection: 200 ng/mL OTHER SUBSTANCES Simultaneous: metabolites
KEYWORDS

plasma; rat; pharmacokinetics

REFERENCE

Ogiso,T.; Iwaki,M.; Tanino,T.; Ikeda,K.; Paku,T.; Horibe,Y.; Suzuki,H. Pharmacokinetics of aniracetam and its metabolites in rats, J.Pharm.ScL, 1998, 87, 594-598.
SAMPLE

Matrix: blood Sample preparation: 500 |xL Plasma + 25 yJu 5 jxg/mL IS in water, mix thoroughly. Maintain sample at 4 until injection, inject 200 JXL plasma onto column A with mobile phase A, after 4 min backflush the contents of column A onto column B with mobile phase B, after 1 min remove column A from the circuit, monitor the effluent from column B. Wash column A with MeOH for 10.8 min, re-equilibrate with water for 10 min. At the end of the separation backflush column A with mobile phase B for 12 min.
HPLC VARIABLES

Column: A 20 X 2.1 30 |xm Hypersil ODS; B 30 X 4.6 10 jim Brownlee RP-2 + 60 x 4 3 |xm Hypersil ODS Mobile phase: A water; B MeOH:MeCN:water 30:10:70 Flow rate: A 2; B 1 Injection volume: 200 Detector: UV 282 CHROMATOGRAM Retention time: 8.3 Internal standard: l-(p-ethoxybenzoyl)-2-pyrrolidinone(Ro 13-8606) (11.8) Limit of quantitation: 5 ng/mL

KEYWORDS

plasma; column switching

REFERENCE Guenzi,A.; Zanetti,M. Determination of aniracetam and its main metabolite, N-anisoyl-GABA, inhuman plasma by high-performance liquid chromatography, J.Chromatogr., 1990, 530, 397-406.

Next Page

Anistreplase
Molecular formula: indeterminate CAS Registry No.: 81669-57-0 Merck Index: 712

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: Bakerbond C4 Mobile phase: Gradient. MeCN:0.1% trifluoroacetic acid from 0:100 to 60:40 over 90 min Flow rate: 1 Injection volume: 200 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: alteplase, streptokinase, urokinase

REFERENCE Werner,R.G.; Bassarab,S.; Hoffmann,H-; Schliiter,M. Quality aspects of fibrinolytic agents based on biochemical characterization, Arzneimittelforschung, 1991, 41, 1196-1200.

Previous Page

Antazoline
Molecular formula: C17H19N3 Molecular weight: 265.36 CAS Registry No.: 91-75-8, 2508-72-7 Merck Index: 718

LednicerNo.: 1 242 SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |JLL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mIVL 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 2.6 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,

protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine chemically bonded to silica (Regis) Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80 Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: k' 11.04
OTHERSUBSTANCES

Also analyzed: acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bopindolol, bupranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cinnarizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, esmolol, famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304
REFERENCE Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on aj-acid glycoprotein as revealed by chemometric analysis of biochromatographic data, Biomed.Chromatogr., 1995, 9, 211-215.

Anthralin
Molecular formula: C14H10O3 Molecular weight: 226.23 CAS Registry No.: 1143-38-0 Merck Index: 723

SAMPLE

Matrix: bulk Sample preparation: Prepare a 1 mg/mL solution in dichloromethane. Mix a 1 mL aliquot with 19 mL dichloromethane and 1 mL acetic acid, make up to 50 mL with hexane. Inject an aliquot.
HPLC VARIABLES

Guard column: 4 X 4 5 jim LiChrospher 100 RP18 Column: 250 X 4 5 |xm LiChrospher 100 RP18 Mobile phase: MeOH:water:acetic acid 77:23:0.1, adjusted to pH 5.5 with cone, ammonia Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 15.6 OTHER SUBSTANCES Simultaneous: impurities
REFERENCE Muller,K.; Ziereis,K.; Wiegrebe,W. The monograph dithranol in the European Pharmacopoeia -comment and amendments, Pharmazie, 1996, 51, 980-981. SAMPLE

Matrix: formulations Sample preparation: Creams, gels, lotions. Disperse and dilute in MeCN to an anthralin concentration of 0.25 mg/mL. Remove a 10 mL aliquot and add it to 10 mL 1 mg/mL anthracene in MeCN, 10 mL chloroform, 20 mL MeCN, and 0.25 mL acetic acid. Filter (Whatman No. 42 paper), inject a 10 |xL aliquot. Sticks, ointments. Disperse and dilute in chloroform to an anthralin concentration of 0.25 mg/mL. Remove a 10 mL aliquot and add it to 10 mL 1 mg/mL anthracene in MeCN, 30 mL MeCN, and 0.25 mL acetic acid. Filter (Whatman No. 42 paper), inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 ixBondapak C18 Mobile phase: MeCN:water:acetic acid 60:39.5:0.5 containing 0.05% sodium hexanesulfonate Flow rate: 2.5 Injection volume: 10 Detector: UV 365
CHROMATOGRAM

Retention time: 6.5 Internal standard: anthracene (8.5) OTHER SUBSTANCES Simultaneous: danthron, dianthrone

KEYWORDS protect from light; deaerate all solvents with argon; creams; gels; lotions; sticks; ointments REFERENCE Burton,F.W.; Gadde,R.R. Analysis of anthralin in dermatological products by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1985, 328, 317-324.

Antipyrine
Molecular formula: C11H12N2O Molecular weight: 188.23 CAS Registry No.: 60-80-0, 520-07-0 (salicylate) Merck Index: 757 Lednicer No.: 1 234 SAMPLE

Matrix: amniotic fluid, blood Sample preparation: Condition a 3 mL Bond Elut C18 SPE cartridge with 2 mL MeOH and 2 mL water. 50 jxL Plasma or amniotic fluid + 50 |xL 5 |xg/mL 3-hydroxyacetamidophenol in water, add to SPE cartridge, wash twice with 2 mL portions of water, elute with 2 mL MeOH. Evaporate the eluate under vacuum, reconstitute the residue in 100 JJLL MeCN:water 6:94, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere ODS C18 Mobile phase: Gradient. A was MeCNrpH 4.0 ammonium phosphate buffer 6:94. B was MeCN:pH 4.0 ammonium phosphate buffer 25:75. A:B from 100:0 to 100:0 over 20 min, to 100:0 over 5 min, re-equilibrate for 10 min. Flow rate: 1 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Limit of quantitation: 400 ng/mL OTHER SUBSTANCES Extracted: didanosine

KEYWORDS

plasma; monkey; pharmacokinetics; SPE

REFERENCE Pereira,C.M.; Nosbisch,C; Winter,H.R.; Baughman,W.L.; Unadkat,J.D. Transplacental pharmacokinetics of dideoxyinosine in pigtailed macaques, Antimicrob.Agents Chemother., 1994, 38, 781-786. SAMPLE

Matrix: amniotic fluid, blood Sample preparation: Condition a 3 mL Bond Elut C18 SPE cartridge with 2 mL MeOH and two 2 mL portions of water. 100 |xL Plasma or amniotic fluid + 20 |xL 10 fjig/mL 3hydroxyacetamidophenol in water, mix, add to the SPE cartridge, wash with two 2 mL portions of water, elute with 2 mL MeOH. Evaporate the eluate to dryness under reduced pressure, reconstitute with 100 |xL MeCN:50 mM pH 3.3 ammonium phosphate buffer 6: 94, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jxm Ultrasphere ODS Mobile phase: Gradient. A was MeCN:50 mM pH 3.3 ammonium phosphate buffer 6:94. B was MeCN:50 mM pH 3.3 ammonium phosphate buffer 25:75. A:B from 100:0 to 0:100 over 17 min, maintain at 0:100 for 5 min, return to initial conditions over 3 min, reequilibrate for 17 min. Flow rate: 1 Injection volume: 50

Detector: UV 266
CHROMATOGRAM

Limit of quantitation: 400 ng/mL OTHER SUBSTANCES Extracted: stavudine

KEYWORDS

monkey; plasma; pharmacokinetics; SPE

REFERENCE Odinecs,A.; Nosbisch,C; Keller,R.D.; Baughman,W.L.; Unadkant,J.D. In vivo maternal-fetal pharmacokinetics of stavudine (2',3'-didehydro-3'-deoxythymidine in pigtailed macaques (Macaca nemestrina), Antimicrob.Agents Chemother., 1996, 40, 196-202. SAMPLE

Matrix: blood Sample preparation: 0.5 mL Plasma + 0.5 mL water + 0.5 mL 0.25 M NaOH, vortex, allow to stand at room temperature for 20 min, add 20 |xL of 100 |ig/mL phenacetin and 3 |xg/mL flunitrazepam, vortex, add 5 mL diethyl ether, vortex for 30 s, centrifuge at 900 g for 5 min, freeze in acetone/dry ice for 5 min. Remove the supernatant and dry it under nitrogen. Reconstitute in 115 JXL MeCNrO. 1% pH 3 sodium phosphate buffer 30:70, inject an aliquot.
HPLC VARIABLES

Guard column: 23 X 3.9 37-50 |xm jxBondapak phenyl Column: 300 X 3.9 10 |xm ixBondapak phenyl Mobile phase: Gradient. A was MeCN:0.1% pH 3 sodium phosphate buffer 5:95. B was MeCNrO. 1% pH 3 sodium phosphate buffer 70:30. A:B 80:20 for 2.5 min, then to 45:55 over 20 min, then to 25:75 over 3 min, then to 80:20 over 3 min, equilibrate at 80:20 for 7 min. Column temperature: 40 Flow rate: 2 Injection volume: 200 Detector: UV 254
CHROMATOGRAM

Retention time: 5.08 Internal standard: phenacetin (7.09) Limit of quantitation: 5500 ng/mL OTHER SUBSTANCES Simultaneous: lorazepam (at 229 nm) Noninterfering: acetaminophen, trimethoprim, sulfamethoxazole, allopurinol, indocyanine green
KEYWORDS

plasma
REFERENCE Riley,C.A.; Evans,W.E. Simultaneous analysis of antipyrine and lorazepam by high-performance liquid chromatography, J.Chromatogr., 1986, 382, 199-205.

SAMPLE Matrix: blood

Sample preparation: 0.5 mL Plasma +10 |xL 250 jxg/mL 1-acetamidopyrene in MeOH + 200 |ULL 1 M ammonium sulfate + 800 JJLL cold MeCN, vortex for 30 s, store at -20 for at least 30 min, vortex, centrifuge at 1500 g for 30 min. Remove 400 (xL of the upper organic layer and evaporate it under a stream of nitrogen. Reconstitute with 100 |xL mobile phase, vortex for 30 s, inject a 75 JJLL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 ^m ixBondapak C18 Mobile phase: MeCNibuffer 47:53 (Buffer was 6.805 g potassium monophosphate in 1 L water, adjust pH to 6.00 with 10 M NaOH.) Flow rate: 1 Injection volume: 75 Detector: UV 214
CHROMATOGRAM

Retention time: 4.2 Internal standard: 1-acetamidopyrene (9.7) Limit of detection: 100 ng/mL
OTHER SUBSTANCES

Simultaneous: lorazepam, indocyanine green Noninterfering: adenosine, albuterol, alphenal, aspirin, caffeine, carbamazepine, cefazolin, cephalexin, cephalothin, cimetidine, ciprofloxacin, claforan, desipramine, enoxacin, fleroxacin, furosemide, hydralazine, hydrochlorothiazide, minoxidil, norfloxacin, phenytoin, propafenone, sulindac, teicoplanin, theophylline, vancomycin Interfering: some indocyanine green impurities
KEYWORDS

plasma
REFERENCE Awni,W.M.; Bakker,L.J. Antipyrine, indocyanine green, and lorazepam determined in plasma by highpressure liquid chromatography, Clin.Chem., 1989, 35, 2124-2126. SAMPLE

Matrix: blood Sample preparation: Make plasma alkaline with NaOH, extract with dichloromethane.
HPLC VARIABLES

Column: reverse phase Mobile phase: MeOH:water 43:57 containing 100 mM triethylamine, adjusted to pH 4.7 with orthophosphoric acid Detector: UV 254
CHROMATOGRAM

Internal standard: phenacetin Limit of detection: 100 ng/mL

KEY WORDS

plasma
REFERENCE Hayton,W.L.; Kneer,J.; Blouin,R.A.; Stoeckel,K. Pharmacokinetics of intravenous cefetamet and oral cefetamet pivoxil in patients with hepatic cirrhosis, Antimicrob.Agents Chemother., 1990, 34, 13181322.

SAMPLE Matrix: blood

Sample preparation: 200 jxL Serum + 200 |xL mobile phase, filter (Millipore Millex 0.45 |xm), inject a 25 jxL aliquot.
HPLC VARIABLES

Guard column: 40 X 4 C18 Corasil II Column: 300 X 4 10 |xm ixBondapak phenyl Mobile phase: Propanol:6 mM C12 DAPS (Fluka) 3:97 (C12 DAPS is 3-(dimethyldodecylammonio) propanesulfonate.) Injection volume: 25 Detector: UV 273
CHROMATOGRAM

Retention time: 8 Internal standard: antipyrine

OTHER SUBSTANCES

Simultaneous: theophylline, P-hydroxytheophylline, caffeine, theobromine, albendazole, albendazole sulfoxide, nimorazole, flubendazole, mercaptopurine, aminophylline, amyleine, procaine Interfering: metronidazole, tinidazole, dipropyline
KEYWORDS

serum; micellar chromatography; antipyrine is IS

REFERENCE Habel,D.; Guermouche,S.; Guermouche,M.H. Direct determination of theophylline in human serum by high-performance liquid chromatography using zwitterionic micellar mobile phase. Comparison with an enzyme multiplied immunoassay technique, Analyst, 1993, 118, 1511-1513. SAMPLE

Matrix: blood Sample preparation: 100 |xL Plasma + 1 ug phenacetin + 100 |xL 100 mM NaOH + 400 |JLL water + 3 mL dichloromethane, shake vigorously for 15 min, centrifuge at 1500 g for 10 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL mobile phase, inject a 40 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 7 urn Hibar LiChrosorb RP-18 Mobile phase: MeCNrIO mM pH 8 phosphate buffer 20:80 Flow rate: 1.5 Injection volume: 40 Detector: UV 254
CHROMATOGRAM

Retention time: 6 Internal standard: phenacetin (12) Limit of detection: <500 ng/mL
KEYWORDS

plasma
REFERENCE Wolfisberg,H.; Schmutz,A.; Stotzer,R.; Thormann,W. Assessment of automated capillary electrophoresis for therapeutic and diagnostic drug monitoring: determination of bupivacaine in drain fluid and antipyrine in plasma, J.Chromatogr.A, 1993, 652, 407-416. SAMPLE Matrix: blood

Sample preparation: 50 jxL Plasma + 500 |xL 370 mM pH 4.6 acetate buffer + cyclobarbital + 5 mL dichloromethane, vortex, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 70 |xL mobile phase, inject an aliquot.
HPLC VARIABLES

Guard column: 30 X 4.6 Cyclobond II (Baker) Column: 250 x 4.6 Cyclobond II (Baker) Mobile phase: MeOH:water 15:85 Flow rate: 0.7 Injection volume: 20 Detector: UV 214
CHROMATOGRAM

Internal standard: cyclobarbital Limit of quantitation: 20 ng/mL

OTHER SUBSTANCES

Extracted: hexobarbital, phenobarbital

KEYWORDS

plasma; rat
REFERENCE Groen,K; Breimer,D.D.; Jansen,E.J.; Van Bezooijen,C.F.A. The influence of aging on the metabolism of simultaneously administered hexobarbital enantiomers and antipyrine before and after phenobarbital induction in male rats: A longitudinal study, J.Pharmacol.Exp.Ther., 1994, 268, 531-536. SAMPLE

Matrix: blood Sample preparation: 100 |xL Plasma + 100 |xL 20 jxg/mL cefadroxil + 8 mL dichloromethane, shake for 20 min, centrifuge at 2500 rpm for 20 min. Remove 7 mL of the organic layer and evaporate it to dryness under nitrogen or at 60. Dissolve residue in 200 |iL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 150 X 6 Shimpack CLS-ODS (Shimadzu) Mobile phase: MeCN:0.5 mM phosphoric acid 12:88 Column temperature: 40 Flow rate: 1.5 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Internal standard: cefadroxil

KEYWORDS

plasma; rat
REFERENCE Lee,C.K.; Uchida,T.; Kitagawa,K.; Yagi,A.; Kim,N.-S.; Goto,S. Skin permeability of various drugs with different lipophilicity, J.Pharm.Sci., 1994, 83, 562-565. SAMPLE

Matrix: blood Sample preparation: 0.5 mL Plasma + 25 J L J L L 40 |xg/mL 3-acetamidophenol extracted with ether:dichloromethane:isopropanol 60:40:1. Evaporate organic layer under a stream of nitrogen and take up residue in 250 |xL 50 mM pH 7.8 phosphate buffer.

HPLC VARIABLES

Column: 100 X 4.6 Chrompack microsphere C18 Mobile phase: Gradient. A was 50 mM pH 7.8 buffer. B was MeOH:50 mM pH 7.8 phosphate buffer 50:50. A:B from 100:0 to 50:50 over 38 min, maintain at 50:50 for 2 min. Flow rate: 0.7 Injection volume: 250 Detector: UV 254
CHROMATOGRAM

Internal standard: 3-acetamidophenol

OTHER SUBSTANCES

Simultaneous: acetaminophen, metabolites

KEYWORDS

plasma; pig
REFERENCE Monshouwer,M.; Witkamp,R.E; Pijpers,A.; Verheijden,J.H.M.; Van Miert,A.S.J.PA.M. Dose-dependent pharmacokinetic interaction between antipyrine and paracetamol in vivo and in vitro when administered as cocktail in pig, Xenobiotica, 1994, 24, 347-355. SAMPLE

Matrix: blood Sample preparation: 0.5 mL Plasma + 25 |xL 40 |ig/mL 3-acetamidophenol extracted with ether:dichloromethane:isopropanol 60:40:1. Evaporate organic layer under a stream of nitrogen and take up residue in 50 mM pH 7.8 phosphate buffer, inject an aliquot.
HPLC VARIABLES

Column: 100 X 4.6 Chrompack microsphere C18 Mobile phase: Gradient. A was 50 mM pH 7.8 phosphate buffer. B was MeOH:50 mM pH 7.8 phosphate buffer 50:50. A:B from 100:0 to 50:50 over 38 min, maintain at 50:50 for 2 min. Flow rate: 0.7 Injection volume: 250 Detector: UV 254
CHROMATOGRAM

Internal standard: 3-acetamidophenol OTHER SUBSTANCES Extracted: acetaminophen

KEYWORDS

plasma; metabolites; pig; pharmacokinetics

REFERENCE Monshouwer,M.; Witkamp,R.E; Pijpers,A.; Verheijden,J.H.M.; Van Miert,A.S.J.P.A.M. Dose-dependent pharmacokinetic interaction between antipyrine and paracetamol in vivo and in vitro when administered as cocktail in pig, Xenobiotica, 1994, 24, 347-355.

SAMPLE Matrix: blood Sample preparation: Plasma + phenacetin + 10% trichloroacetic acid, centrifuge, inject a
20 J L J L L aliquot of the supernatant.

HPLC VARIABLES

Column: 100 X 4.6 3 ^m Nucleosil C18 Mobile phase: MeCN:buffer 25:75 (Buffer was 50 mM citrate adjusted to pH 5 with acetic acid.) Column temperature: 50 Flow rate: 1.5 Injection volume: 20 Detector: UV 244
CHROMATOGRAM

Internal standard: phenacetin Limit of quantitation: 500 ng/mL

KEYWORDS

dog; plasma; pharmacokinetics

REFERENCE Galtier,M.; Briand,D.; Pinguet,R; Gomeni,R.; Fabre,D.; Bressolle,F. Pharmacokinetic parameters of antipyrine in dog after hepatectomy, Biopharm.Drug Dispos., 1995, 16, 669-684. SAMPLE

Matrix: blood, saliva Sample preparation: 1 mL Plasma or saliva + 100 jxL 400 [ig/mh phenacetin in EtOH + 100 jxL 2 M NaOH + 5 mL dichloromethanern-pentane 50:50, vortex for 15 s. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 100 \LL mobile phase, inject a 25 |xL aliquot.
HPLC VARIABLES

Column: 10 |xm Spheri-10 RP-18 Mobile phase: MeCN:100 mM sodium acetateitriethylamine 7.5:92:0.5, pH 6.6 Column temperature: 40 Flow rate: 3.5 Injection volume: 25 Detector: UV 254
CHROMATOGRAM

Internal standard: phenacetin

KEYWORDS

plasma; pharmacokinetics
REFERENCE Jorquera,E; Almar,M.M.; Jimeno,A.; Gonzalez-Sastre,M.; Gonzalez-Gallego,J. Assessment of antipyrine kinetics from saliva or plasma: influence of age, JJPharm.Biomed.Anal., 1995, 13, 1141-1145. SAMPLE

Matrix: blood, urine Sample preparation: Plasma. Mix 500 |xL plasma with 25 |xL 15 jxg/mL IS in MeOH, 500 IxL 750 mM pH 5.0 sodium acetate buffer containing 40 mg/mL sodium metabisulfite, and 10 mg of a mixture of p-glucuronidase and arylsulfatase (Limpet acetone powder Type I: Platela vulgata, Sigma). Heat at 37 for 2 h, add 100 mg NaCl, extract with 1.2 mL chloroform:EtOH 90:10 for 2 min (Caution! Chloroform is a carcinogen!). Centrifuge at 1800 g for 20 min, add the organic phase to 50 |xL 100 mM HCl, evaporate to dryness under a stream of nitrogen, dissolve the residue in 75 jxL mobile phase, wash with 25 |xL n-hexane, inject a 20 |xL aliquot of the lower phase. Urine. Mix 500 |xL urine with 25 |xL 500 |xg/mL IS in MeOH, 500 (xL 750 mM pH 5.0 sodium acetate buffer containing 40 mg/ mL sodium metabisulfite, and 10 mg of a mixture of p-glucuronidase and arylsulfatase

(Limpet acetone powder Type I: Platela vulgata, Sigma). Heat at 37 for 2 h, add 200 mg sodium chloride, extract with 2 mL dichloromethane:isopropanol 90:10 for 90 s, centrifuge at 1800 g for 30 min, evaporate the organic phase under a stream of nitrogen. Dissolve the residue in 200 |xL mobile phase, vortex for 15 s, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 150 X 3.9 Nova-Pak C18 Mobile phase: MeOH:250 mM pH 5.0 sodium acetate buffer 30:70 Flow rate: 1.0 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 4.5 Internal standard: phenacetin (10) OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

plasma; pharmacokinetics
REFERENCE Lanchote,V.L.; Ping,W.C; Santos,S.R.C.J. Determination of antipyrine and metabolites in plasma of a patient with mild renal failure, Ther.Drug Monit, 1997, 19, 705-710. SAMPLE

Matrix: blood, urine Sample preparation: 1 mL Plasma or urine + 500 jxL 100 mM NaOH + 10 mL benzene (CAUTION! Benzene is a carcinogen!), shake vigorously for 20 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 250 \iL MeOH, inject a 25 jxL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 (xm ixBondapak C18 Mobile phase: MeOH:water:acetic acid 20:75:5 Flow rate: 1 Injection volume: 25 Detector: UV 254 for 7 min then UV 280 CHROMATOGRAM Retention time: 8.0 Internal standard: antipyrine
OTHER SUBSTANCES

Extracted: 4-monomethylaminoantipyrine
KEYWORDS

plasma; antipyrine is IS
REFERENCE Asmardi,G.; Jamali,R High-performance liquid chromatography of dipyrone and its active metabolite in biological fluids, J.Chromatogr., 1983, 277, 183-189. SAMPLE

Matrix: saliva Sample preparation: Add 100 |xL phenacetin to 1 mL saliva, inject an aliquot.

HPLC VARIABLES

Column: 10 |xm Spheri-10 RP-18 (Brownlee) Mobile phase: MeCNrbufFer 7.5:92.5 (The buffer was 100 mM sodium acetate containing 0.5% triethylamine, pH 6.6.) Column temperature: 40 Flow rate: 3.5 Detector: UV 254
CHROMATOGRAM

Internal standard: phenacetin

KEY WORDS

pharmacokinetics; saliva
REFERENCE Jorquera,E; Almar,M.M.; Gonzalez-Sastre,M.; Suarez,L; Gonzalez-Gallego,J. Accuracy of the one-sample method for determination of antipyrine clearance in elderly subjects, J.Pharm.Biomed.AnaL, 1996, 15, 7-11. SAMPLE

Matrix: saliva Sample preparation: 500 |xL Saliva + 500 |xL MeCN + 50 |xL 400 jjig/mL phenacetin, vortex, centrifuge at 5500 g for 1 min. Filter (4.5 jjim) the supernatant and inject a 20 jxL aliquot of the supernatant.
HPLC VARIABLES

Column: 250 X 4.6 7 |jim Eicopack MA-ODS (Eicom, Kyoto) Mobile phase: MeCN:20 mM pH 6.0 potassium phosphate buffer 30:70 containing 0.1% triethylamine Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 4.5 Internal standard: phenacetin (8.2) Limit of detection: 100 ng/mL
REFERENCE Echizen,H.; Nakura,M.; Ishizaki,I. Rapid and simple high-performance liquid chromatographic determination of saliva antipyrine for routine antipyrine test, J.Chromatogr., 1990, 526, 296-299. SAMPLE

Matrix: saliva Sample preparation: 1 mL Saliva + 100 |xL 2 M NaOH + 100 uL 400 (xg/mL phenacetin in EtOH + 5 mL n-pentane:dichloromethane 50:50, extract on a whirlmixer for 15 s. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 100 U | LL mobile phase, inject a 25 |xL aliquot.
HPLC VARIABLES

Column: 10 |xm Spheri-10 RP-18 Mobile phase: MeCN:100 mM sodium acetate:triethylamine 7.5:92:0.5, pH 6.6 Column temperature: 40 Flow rate: 3.5 Injection volume: 25 Detector: UV 254

CHROMATOGRAM

Internal standard: phenacetin

KEYWORDS

pharmacokinetics
REFERENCE Jorquera,E; Almar,M.M.; Pozuelo,M.; Sansegundo,D.; Gonzalez-Sastre,M.; Gonzalez-Gallego,J. Effects of aging on antipyrine clearance: Predictive factors of metabolizing capacity, J.Clin.PharmacoL, 1995, 35, 895-901.

SAMPLE Matrix: saliva Sample preparation: Saliva + phenacetin + MeCN, vortex, centrifuge, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 100 X 4.6 5 |xm ODS-Technosphere (HPLC Technology) Mobile phase: MeCN:50 mM pH 6.0 sodium phosphate buffer 1:2 Flow rate: 1,5 Injection volume: 20 Detector: UV 260
CHROMATOGRAM

Internal standard: phenacetin Limit of detection: 10OnM

REFERENCE Perrett,D.; Ross,G.A. Rapid determination of drugs in biofluids by capillary electrophoresis. Measurement of antipyrine in saliva for pharmacokinetic studies, J.Chromatogr.A, 1995, 700, 179-186. SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 250 X 4 ODS (Hitachi) Mobile phase: MeCN:50 mM phosphoric acid 40:60 containing 400 mM KCl Column temperature: 55 Flow rate: 0.6 Injection volume: 20 Detector: UV 230
OTHERSUBSTANCES

Also analyzed: chlorpheniramine

REFERENCE Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K.; Miyazaki,K. A general approach for the prediction of the intestinal absorption of drugs: regression analysis using the physicochemical properties and drug-membrane electrostatic interactions, J.Pharm.ScL, 1998, 87, 960-966. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH.water 1:1 at a concentration of 50 jjug/mL, inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 |im jxBondapak C18

Mobile phase: MeOH:acetic acid:triethylamine:water 30:1.5:0.5:68 Flow rate: 1.5 Injection volume: 10 Detector: UV CHROMATOGRAM Retention time: k' 2.60
REFERENCE Roos,R-W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazo-

cine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994,18,233-242. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 jjim Supelcosil LC-DP (A) or 250 X 4 5 jjim LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 niL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 5.39 (A), 4.54 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,

phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 \xL aliquot of an aqueous solution.

HPLC VARIABLES

Column: 150 X 3 4 jim Supersphere 100 RP18 endcapped (Bischoff) Mobile phase: MeCN:20 mM pH 7.4 KH2PO4 10:90 Flow rate: 0.5 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 25
OTHER SUBSTANCES

Simultaneous: degradation products, radiation degradation products, o-hydroxyantipyrine, m-hydroxyantipyrine, p-hydroxyantipyrine

KEYWORDS

comparison with capillary electrophoresis

REFERENCE Coolen,S.A.J.; Everaerts,F.M.; Huf,F.A. Characterization Of60Co 7-radiation induced radical products of antipyrineby means of high-performance liquid chromatography, mass spectrometry, capillary zone electrophoresis, micellar electrokinetic capillary chromatography and nuclear magnetic resonance spectrometry, J.ChromatogrA, 1997, 788, 95-103. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 jxL aliquot of a 100-500 jig/mL solution in mobile phase.
HPLCVARIABLES

Column: 100 X 4.6 5 jjim Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure soybean lecithin, Epikuron, Lucas Meyer & Co.) (Coat column by recycling a 1 mM solution of phosphatidylcholine in MeOHrwater 80:20 for 24 h.) Mobile phase: MeCN:35 mM pH 7.4 sodium phosphate buffer 40:60 Flow rate: 0.5-2 Injection volume: 20 Detector: UV 254

CHROMATOGRAM Retention time: k' 1.70

OTHER SUBSTANCES

Also analyzed: amoxicillin, carbamazepine, chlorpheniramine, chlorpromazine, clonidine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, flufenamic acid, haloperidol, hydroxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone, nadolol, phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole, testosterone, thioridazine, tolfenamic acid, verapamil Noninterfering: acetaminophen, aspirin, azathioprine, caffeine, carprofen, chlorambucil, cimetidine, fenoterol, flurbiprofen, ibuprofen, ketoprofen, ranitidine, salicylic acid, sulfamethoxazole, theophylline, thioguanine, tiaprofenic acid, trimethoprim, valproic acid
KEYWORDS

comparison with capillary electrophoresis

REFERENCE Hanna,M.; de Biasi,V.; Bond,B.; Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics of drugs: A comparison of a large and diverse drug series utilizing chromatographic and electrophoretic methodology, Anal.Chem., 1998, 70, 2092-2099. SAMPLE

Matrix: urine Sample preparation: Condition a C18 AASP SPE cartridge (Varian) with 1 mL MeOH and
1 mL water. 100 JJIL Urine + 100 JULL 50 mM pH 4.5 KH2PO4 + 20 mg limpet acetone

powder (type I, contains p-glucuronidase and sulfatase, from Sigma), + 16 mg sodium metabisulfite, vortex for 15 s, heat at 40 with mild agitation for 3 h. Cool to room temperature, add 10 |xL 1 mg/mL 4-dimethylaminoantipyrine in water, vortex, centrifuge at 500 g for 20 min. Remove 100 JJLL supernatant and add it to 900 jxL pH 4.5 100 mM TRIS. Remove 500 JJLL and add it to 500 |xL 50 mM pH 4.5 KH2PO4, add to SPE cartridge, wash with four 1 mL portions of 50 mM pH 4.5 phosphate buffer, allow to dry completely, elute with three 100 |xL volumes of MeCN:dichloromethane 20:80. Evaporate eluate at 40 under vacuum while vortexing, reconstitute the residue in 100 JULL 50 mM pH 4.5 phosphate buffer, inject a 20 |xL aliquot.
HPLC VARIABLES

Guard column: 20 X 2 40 jxm Corasil phenyl Column: 100 X 4.6 5 |xm Spherisorb phenyl Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeOH. A:B from 90:10 to 60:40 over 14 min, to 40:60 over 7 min, to initial conditions over 0.1 min, re-equilibrate for 7.9 min. Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 17.8 Internal standard: 4-dimethylaminoantipyrine (19.9) Limit of quantitation: 2000 ng/mL OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

SPE

REFERENCE Sarkar,M.A.; March,C; Karnes,H.T. Solid phase extraction and simultaneous high performance liquid chromatographic determination of antipyrine and its major metabolites in urine, Biomed.Chromatogr., 1992, , 300-304. SAMPLE Matrix: urine Sample preparation: Inject a 100 |xL aliquot directly. HPLCVARIABLES Guard column: 25 X 4 5 urn LiChrospher 100 RP 18 Column: 125 X 4 5 jim LiChrospher 60 RP Select B Mobile phase: Gradient. A was 5 mM tetrabutylammonium phosphate in water. B was 5 mM tetrabutylammonium phosphate in MeOH. A:B from 90:10 to 87:13 over 10 min, to 67:33 over 16 min, to 60:40 over 9 min, to 0:100 over 10 min, maintain at 0:100 for 5 min, return to initial conditions over 5 min. Flow rate: 0.7 Injection volume: 100 Detector: UV 254 CHROMATOGRAM Retention time: 23 OTHER SUBSTANCES Extracted: metabolites KEYWORDS rat; dog REFERENCE Velic,L; Metzler,M.; Hege,H-(j.; Weymann,J. Separation and identification of phase I and phase II [14C]antipyrine metabolites in rat and dog urine, J.Chromatogr.B, 1995, 666y 139-147.

Antitrypsin
Molecular formula: indeterminate CAS Registry No.: 9041 -92-3 Merck Index: 760 SAMPLE

Matrix: blood Sample preparation: Dialyze 2 mL plasma against 30 mM pH 7.0 sodium phosphate buffer overnight, chromatograph on 20 mL Blue-Sepharose (equilibrated with 30 mM pH 7.0 sodium phosphate buffer) at 40 mL/h. Dialyze non-retained fraction (20-30 mL) against 30 mM pH 5.8 sodium acetate buffer overnight, chromatograph on 10 mL Red-Sepharose (equilibrated with 30 mM pH 5.8 sodium acetate buffer). Concentrate non-retained fraction (30-50 mL) to 2-3 mL with Amicon PM-10 and freeze dry, dissolve freeze-dried sample in 300 |xL 10 mM pH 7.0 sodium phosphate buffer containing 100 mM NaCl, inject into HPLC.
HPLC VARIABLES

Column: 100 X 6 KB-column hydroxy-apatite (Koken) Mobile phase: Gradient. A was 10 mM pH 7.0 sodium phosphate buffer containing 100 mM NaCl. B was 200 mM pH 7.0 sodium phosphate buffer containing 100 mM NaCl. A: B from 100:0 to 50:50 over 1 h. Flow rate: 1 Detector: UV 280
CHROMATOGRAM Retention time: 22.3

OTHER SUBSTANCES Extracted: al-acid glycoprotein

KEYWORDS

plasma
REFERENCE Funae,Y.; Wada,S.; Imaoka,S.; Hirotsune,S.; Tominaga,M.; Tanaka,S.; Kishimoto,T.; Maekawa,M. Chromatographic separation of al-acid glycoprotein from al-antitrypsin by high-performance liquid chromatography using a hydroxyapatite column, J.Chromatogn, 1986, 381, 149-152. SAMPLE

Matrix: blood Sample preparation: Prepare concentrate from plasma (procedure given).
HPLC VARIABLES

Column: superose-12 (Pharmacia) Mobile phase: 500 mM NaCl Flow rate: 0.5 Injection volume: 50 Detector: UV 280
KEYWORDS

plasma
REFERENCE Burnouf,T.; Constans,J.; Clerc,A.; Descamps,J.; Martinache,L.; Goudemand,M. Biochemical and biological properties of an ai-antitrypsin concentrate, Vox Sang., 1987, 52, 291-297.

SAMPLE

Matrix: solutions Sample preparation: Dissolve in PBS.

HPLC VARIABLES

Column: 600 X 7.5 Spheragel-TSK 3000 SW Mobile phase: PBS Flow rate: 1 Injection volume: 20 Detector: UV 230 CHROMATOGRAM Retention time: 16.4 OTHER SUBSTANCES Simultaneous: aggregates
REFERENCE Glaser,C.B.; Busby,T.R; Ingham,K.C; Childs,A- Thermal denaturation of a 1-protease inhibitor. Stabilization by neutral salts and sugars, Am.Rev.Respir.Dis., 1983, 128, 77-81.

Apigenin
Molecular formula: C15H10O5 Molecular weight: 270.24 CAS Registry No.: 520-36-5 Merck Index: 773

SAMPLE

Matrix: tissue Sample preparation: Homogenize epidermal cells in absolute EtOH. Evaporate the solvent, dissolve the residue in 100 |xL absolute EtOH containing 2.4 |xg/mL IS. Filter (0.20 |xm nylon membrane), inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 250 X 2.1 Alltima C18 (Alltech) Mobile phase: MeCN:water 48:52 containing 0.1% trifluoroacetic acid Flow rate: 0.3 Injection volume: 5 Detector: UV 337
CHROMATOGRAM

Retention time: 3.76 Internal standard: quercetin (3.06) Limit of detection: 1.15 ng
KEY WORDS

epidermal cells; mouse

REFERENCE Li,B.; Robinson,D.H.; Birt,D.F. Evaluation of properties of apigenin and [G-3H]apigenin and analytic method development, J.Pharm.ScL, 1997, 86, 721-725.

Apomorphine
Molecular formula: C17H17NO2 Molecular weight: 267.33 CAS Registry No.: 58-00-4, 41372-20-7 (HCI) Merck Index: 787

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 206.4
CHROMATOGRAM Retention time: 8.962 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763, 149-163.

Apramycin
Molecular formula: C21H41N5O11 Molecular weight: 539.58 CAS Registry No.: 37321-09-8 Merck Index: 792

SAMPLE

Matrix: fermentation solutions Sample preparation: 5 mL Fermentation broth + 5 mL saturated aqueous solution of Tris + 20 mL MeCN, centrifuge at 3000 rpm for 10 min. Remove a 1 mL aliquot of the supernatant and add it to 3 mL 150 mM 2,4-dinitrofluorobenzene in MeOH, heat at 100 under a reflux condenser for 45 min, make up to 4 mL with mobile phase, inject an aliquot.
HPLC VARIABLES

Column: 200 X 4.6 10 |xm LiChrosorb RP-8 Mobile phase: MeCN:water:acetic acid 55:45:0.15 Flow rate: 1.2 Injection volume: 20 Detector: UV 350
CHROMATOGRAM Retention time: 9.56 OTHER SUBSTANCES

Extracted: kanamycin B, tobramycin

KEYWORDS

derivatization
REFERENCE Harangi,J.; Deak,M.; Nanasi,R; Bacsa,G. Determination of the major factors of fermentation of the nebramycin complex by high performance liquid chromatography, J.Liq.Chromatogr., 1984, 7, 8 3 93. SAMPLE

Matrix: reaction mixtures Sample preparation: 50 \xL Buffered reaction mixture + 50 |xL isopropanol + 50 |xL reagent, heat at 60 for 10 min, centrifuge at 1000 g for 2 min, immediately inject a 50 |JLL aliquot of the supernatant. (Reagent was 80 mM o-phthalaldehyde and 250 mM thioglycolic acid in 1 M boric acid, pH adjusted to 10.4 with 40% KOH.)
HPLC VARIABLES

Column: 100 X 5 Hypersil ODS Mobile phase: A was MeOH:water:acetic acid 50:45:5 containing 5 g/L heptanesulfonic acid. B was MeOH:water:acetic acid 75:20:5 containing 5 g/L heptanesulfonic acid. A:B 70:30. Flow rate: 2 Injection volume: 50 Detector: UV 330
CHROMATOGRAM
Retention time: 20.5

REFERENCE Lovering,A.M.; White,L.O.; Reeves,D.S. Identification of aminoglycoside-acetylating enzymes by highpressure liquid chromatographic determination of their reaction products, Antimicrob.Agents Chemother., 1984,26, 10-12.

Aprindine
Molecular formula: C22H30N2 Molecular weight: 322.49 CAS Registry No.: 37640-71-4, 33237-74-0 (HCI) Merck Index: 793 SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Ibxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 16.967 KEYWORDS whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Aprobarbital
Molecular formula: C10H14N2O3 Molecular weight: 210.23 CAS Registry No.: 77-02-1 Merck Index: 794 LednicerNo.: 1 268

SAMPLE

Matrix: blood Sample preparation: Extract 20 |xL with three 20 JJLL portions of acetonerdiethyl ether 50: 50. Combine the organic layer s and add 5 |xL ethyl acetate, dry over 4 A molecular sieve, evaporate to about 5 |xL (mostly ethyl acetate), add 15 |xg dansyl chloride, add 2 mg potassium carbonate, reflux for 2 h, dilute to 100 |xL, inject a 5 jxL aliquot.
HPLC VARIABLES

Column: 500 X 2.5 30 jxm pellicular C18 Mobile phase: Gradient. MeOH.water 0:100 for 8 min, to 65:35 over 20 min. Flow rate: 1 Injection volume: 5 Detector: F ex 360 em 520 CHROMATOGRAM Retention time: 16 OTHERSUBSTANCES Extracted: barbital, heptabarbital
KEYWORDS derivatization; whole blood REFERENCE Diinges,W.; Naundorf,G.; Seiler,N. High pressure liquid chromatographic analysis of barbiturates in the picomole range by fluorometry of their DANS-derivatives, J.Chromatogr.ScL, 1974, 12, 655-657.

SAMPLE Matrix: blood Sample preparation: Mix plasma with an equal volume of MeCN, centrifuge at 10000 g, dilute supernatant with an equal volume of water, inject a 50 jxL aliquot.
HPLC VARIABLES

Column: 110 X 4.7 5 |xm PartiSphere C18 (Whatman) Mobile phase: MeCN: 15 mM pH 7.0 phosphate buffer 30:70 Flow rate: 0.8 Injection volume: 50 Detector: UV 270 following post-column reaction. The column effluent flowed through a 6 m X 0.25 mm ID crocheted coil of PTFE tubing irradiated by an 8 W low-pressure mercury lamp to the detector. CHROMATOGRAM Retention time: 4.4
OTHER SUBSTANCES

Extracted: butethal, pentobarbital, secobarbital

KEY WORDS

plasma; post-column reaction; post-column photochemical derivatization

REFERENCE Wolf,C; Schmid,R-W. Enhanced UV-detection of barbiturates in HPLC analysis by on-line photochemical reaction, J.Liq.Chromatogr., 1990, 13, 2207-2216. SAMPLE

Matrix: solutions Sample preparation: Prepare a 0.5 mg/mL solution in MeOH, inject a 5 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 150 mM phosphoric acid and 50 mM triethylamine. B was MeCNrwater 80:20 containing 150 mM phosphoric acid and 50 mM triethylamine. A:B 100:0 for 2.2 min then to 0:100 over 30 min. Column temperature: 30 Flow rate: 2 Injection volume: 5 Detector: UV 210 CHROMATOGRAM Retention time: 14.3
OTHER SUBSTANCES

Simultaneous: acetaminophen, butabarbital, chlordiazepoxide, chloroxylenol, chlorpromazine, clenbuterol, cortisone, danazol, diflunisal, estrone, fluoxymesterone, mefenamic acid, methyltestosterone, nicotine, oxazepam, phentermine, phenylpropanolamine, progesterone, sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypromine, tripelennamine Interfering: doxapram
KEYWORDS

details for purification of triethylamine in paper

REFERENCE Hill,D.W.; Kind,A.J. The effects of type B silica and triethylamine on the retention of drugs in silica based reverse phase high performance chromatography, J.Liq.Chromatogr., 1993, 16, 3941-3964. SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 300 X 3.9 ixBondapak C18 Mobile phase: MeCNtIO mm KH2PO4 + 5 mM 1-decanesulfonic acid 30:70, adjusted to pH 3.2 with 85% phosphoric acid Flow rate: 1 Injection volume: 10 Detector: UV 214
CHROMATOGRAM

Retention time: 5.8 Internal standard: methyl paraben (7.0) Limit of detection: 100 ng/mL
OTHER SUBSTANCES

Simultaneous: allobarbital, barbital, butalbital, mephobarbital, pentobarbital, phenobarbital, secobarbital, talbutal, vinbarbital

KEYWORDS

stability-indicating
REFERENCE Ibrahim,RB. Simultaneous determination and separation of several barbiturates and analgesic products by ion-pair high-performance liquid chromatography, J.Liq.Chromatogr., 1993, 16, 2835-2851. SAMPLE

Matrix: solutions Sample preparation: Dissolve in mobile phase to a concentration of 50 jxg/mL.

HPLC VARIABLES

Column: 250 X 4 p-cyclodextrin polymer-coated silica (Chromatographia 1993, 36, 373) Mobile phase: MeOHrwater 50:50 Flow rate: 0.6 Injection volume: 20 Detector: UV 240 CHROMATOGRAM Retention time: k' 0.398
OTHER SUBSTANCES

Simultaneous: pentobarbital, amobarbital, butabarbital, butalbital, secobarbital, thiopental, phenobarbital

REFERENCE Forgacs,E.; Cserhati,T. Retention behaviour of barbituric acid derivatives on a p-cyclodextrin polymercoated silicon column, J.ChromatogrA, 1994, 668, 395-402. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 x 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine,

eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994,18, 233-242.

Aprotinin
Molecular formula: C284H432N84O79S7 Molecular weight: 6511.53 CAS Registry No.: 9087-70-1 Merck Index: 796

SAMPLE

Matrix: blood, urine Sample preparation: Prepare an affinity column with porcine pancreatic kallikrein bound to cyanogen bromide-activated Sepharose 4B according to the method of the manufacturer (Pharmacia). Use prolonged washing cycles of high and low pH. Immediately acidify urine to pH 2.0. Adjust pH of plasma to 8.3 with 100 mM pH 8.3 Tris-HCl buffer containing 500 mM NaCl. Adjust pH of urine to 8.3 with 2 M NaOH. Centrifuge sample at 4390 g for 10 min, pump onto affinity column, pump eluate twice onto column. Wash column with ten bed volumes 100 mM pH 8.3 Tris-HCl buffer containing 500 mM NaCl. Elute with 800 |xL 1 M phosphoric acid and 800 JJLL 10 mM pH 2.2 phosphoric acid in 200 mM sodium perchlorate, mix, inject a 1000 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 7 fim LiChrosorb RP-18 Mobile phase: MeCN:200 mM pH 2.2 sodium perchlorate containing 10 mM phosphoric acid 30:70 Column temperature: 25 Flow rate: 1 Injection volume: 1000 Detector: UV 200
CHROMATOGRAM

Retention time: 9.3 Limit of detection: 50 ng/mL

KEYWORDS

plasma; affinity chromatography

REFERENCE Raspi,G.; Lo Moro,A.; Spinetti,M. High-performance liquid chromatographic method for the determination of aprotinin in body fluids, J.Chromatogr.y 1990, 525, 426-432. SAMPLE

Matrix: formulations Sample preparation: Dilute (if necessary) to a concentration of 500 (xg/mL, inject a 20 u,L aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Supelco LC-304 (pore size 30 nm) Mobile phase: MeCN:31 mM sodium perchlorate 22:78 Flow rate: 0.5 Injection volume: 20 Detector: UV 214
CHROMATOGRAM

Retention time: 35 Limit of detection: 5000 ng/mL

REFERENCE Dimov,N.; Simeonov,S. Purity evaluation of aprotinin by high performance liquid chromatography, Biomed.Chromatogr., 1993, 7, 146-148. SAMPLE

Matrix: solutions Sample preparation: Inject a 100-1000 |xL aliquot of buffer solution containing aprotinin.
HPLC VARIABLES

Column: 250 X 4.6 7 jim LiChrosorb RP-18 Mobile phase: MeCN: 10 mM phosphoric acid in 200 mM sodium perchlorate 30:70 Column temperature: 25 Flow rate: 1 Injection volume: 100-1000 Detector: UV 200
CHROMATOGRAM

Retention time: 9.3 Limit of detection: 600 ng/mL

REFERENCE Raspi,G.; Lo Moro,A.; Spinetti,M.; Molinari,M. Determination of aprotinin by titration with bovine trypsin with end-point detection by high-performance liquid chromatography, Analyst, 1989, 114, 10171019. SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: Vydac C4 (code 214TP54) Mobile phase: Gradient. A was 0.1% trifluoroacetic acid and 2% ammonium sulfate. B was 0.07% trifluoroacetic acid in MeCN. A:B from 95:5 to 75:25 over 20 min. Flow rate: 1.5 Detector: UV 220 CHROMATOGRAM Retention time: 15.8 OTHER SUBSTANCES Simultaneous: degradation products
KEYWORDS cow REFERENCE Vinther,A.; Bjorn,S.E.; Sorensen,H.H.; Soeberg,H. Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry, J.Chromatogr., 1990, 516, 175-184.

Arbaprostil
Molecular formula: C2IH34O5 Molecular weight: 366.50 CAS Registry No.: 55028-70-1 Merck Index: 808 Lednicer No.: 3 8

SAMPLE

Matrix: blood Sample preparation: Condition a 3 mL 200 mg and a 1 mL 100 mg Bond-Elut C18 SPE cartridge with 4 mL MeOH and 4 mL water. 3 mL Plasma + 12 |xL 32.5 ng/mL IS in MeCN + 300 |xL 5% formic acid, vortex, centrifuge at 4 at 1500 g for 15 min, add the supernatant to the 3 mL SPE cartridge, wash with two 2 mL portions of water, wash with two 2 mL portions of MeOH:water 10:90, wash with 2 mL toluene, elute with 1 mL ethyl acetate. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute with 610 JJIL MeOH, vortex for 30 s, add 1.42 mL 0.01% formic acid, inject a 1.8 mL aliquot onto a 30 X 4.6 5 |xm Brownlee RP-8 column and elute to waste with MeCN: water:formic acid 40:60:0.01 at 2 mL/min, after 2.5 min backflush the contents of this column onto a 250 X 4.6 5 |xm Supelcosil LC-18 column eluted with MeCN:water:formic acid 40:60:0.01 at 2 mL/min, after about 6.5-7 min collect a fraction containing the prostaglandins. Dilute this fraction with an equal volume of water, add to the 1 mL SPE cartridge, wash with 1 mL hexane, elute with two 500 jxL portions of ethyl acetate. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 250 |xL 100 |xg/mL panacyl bromide in THF:MeCN 20:80, vortex for 30 s, add 3 |JLL N,Ndiisopropylethylamine, heat at 40 for 1 h (Anal. Chem. 1984, 56, 1866). Evaporate to dryness under a stream of nitrogen at 40, reconstitute the residue in 230 jxL isooctane: ethylene dichloride:isopropanol 70:30:1, sonicate for 10 min, inject a 200 |xL aliquot onto column A and elute to waste with mobile phase A, after 3 min divert the effluent from column A onto column B and elute both to waste, after 1.5 min remove column A from the circuit, continue to elute column B to waste with mobile phase A, after 7.5 min collect the effluent from column B in a 2.2 mL sample loop, after 2 min inject the contents of this sample loop onto column C with mobile phase B, elute column C with mobile phase B, monitor the effluent from column C. (Synthesize panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) as follows. Add 3.04 g benzyltrimethylammonium dichloroiodate to a solution of 500 mg 4'-hydroxyacetophenone in 50 mL dichloroethane and 20 mL MeOH, reflux for 10 h, remove the solvent by distillation, add 20 mL 5% sodium bisulfite to the residue, extract four times with 40 mL portions of ether, dry over anhydrous magnesium sulfate, evaporate to dryness under reduced pressure to give p-hydroxyphenacyl chloride (mp 151-152) (Synthesis 1988, 545). Purify p-hydroxyphenacyl chloride by suspending 100 g in 1 L boiling toluene, filter, cool to obtain white crystals of p-hydroxyphenacyl chloride. Repeat this process a number of times to obtain more pure product. Reflux 10 g 9-anthracenecarboxylic acid in 150 mL redistilled thionyl chloride for 2 h, evaporate to dryness under reduced pressure at 30, dissolve the residue in 150 mL dry toluene containing 11.5 g p-hydroxyphenacyl chloride, reflux for 2 h, evaporate to dryness under reduced pressure, recrystallize from 200 mL hot MeCN to give p-(9-anthroyloxy)phenacyl chloride as deep yellow crystals (mp 159.8-161.6). Dissolve 2.5 g p-(9-anthroyloxy)phenacyl chloride in 25 mL THF.MeCN 20:80, add 8 g anhydrous LiBr, reflux briefly, cool to room temperature, filter, wash the solid with water to obtain p-(9-anthroyloxy)phenacyl bromide as deep yellow crystals (mp 173.3-173.6) (Anal. Biochem. 1987, 165, 220).)
HPLC VARIABLES

Column: A 10 X 4.6 Co:Pell PAC; B 150 X 4.6 6 jim Zorbax CN; C 240 X 4.6 6 |xm Zorbax SiI

Mobile phase: AHexane:dichloromethane:isopropanol 70:30:1; B Hexane:dichloromethane: THF:isopropanol 60:20:20:1 Flow rate: 1 Injection volume: 200 Detector: F ex 375 em 470
CHROMATOGRAM

Retention time: 36 (arbaprostil), 39.5 (15S epimer) Internal standard: 5,6-trans-(15R)-15-methylprostaglandinE2 (U-67205) (37.5) Limit of quantitation: 10 pg/mL
KEYWORDS

derivatization; SPE; plasma; column-switching; normal phase

REFERENCE Pullen,R.H.; Cox,J.W. Determination of (15#)- and (15S)-15-methylprostaglandin E2 in human plasma with picogram per milliliter sensitivity by column-switching high-performance liquid chromatography, J.Chromatogr., 1985, 343, 271-283. SAMPLE

Matrix: formulations Sample preparation: 1 mL Formulation + IS + 500 jxL 2% phosphoric acid, mix, add 10 m l ethyl etherxhloroform 80:20, extract, centrifuge. Remove 8 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 1 mL 2.5 mg/mL p-nitrophenacyl bromide in MeCN, add 500 uL 12.5 |xL/mL N,Ndiisopropylethylamine in MeCN, vortex briefly, heat at 40 for 30 min, evaporate to dryness under a stream of nitrogen at 40, reconstitute the residue in 2 mL mobile phase, vortex, inject a 5-25 |xL aliquot.
HPLCVARIABLES

Column: ixPorasil silica gel Mobile phase: MeCN:dichloromethane:water 30:70:0.5 Flow rate: 1.5 Injection volume: 5-25 Detector: UV 254
CHROMATOGRAM

Retention time: 6.8 Internal standard: 17p-hydroxy-17-methyl-4-androstene-3,ll-dione (5.8) OTHER SUBSTANCES Simultaneous: s-epimer
KEYWORDS

derivatization; injections; normal phase; siliconise glassware with Surfasil (Pierce)

REFERENCE Peng,G.W.; Sood,V.K. Liquid chromatographic assay of arbaprostil, J.Liq.Chromatogr., 1983, 6, 14991511.

Arbidol
Molecular formula: C22H25BrN2O3S Molecular weight: 477.42 CAS Registry No.: 131707-25-0, 131707-23-8 (HCI)

SAMPLE

Matrix: blood Sample preparation: Vortex thawed plasma for 15 s, centrifuge atl4 000 rpm for 1 min. Add 250 |JLL saturated sodium hydrogen carbonate to 1 mL plasma. Add 100 |xL 5 |xg/mL IS in MeCN and 5 mL MTBE. Shake at 50 rpm for 10 min and centrifuge at 4000 rpm for 10 min. Evaporate organic phase at 35 under a gentle flow of nitrogen. Reconstitute the residue in 200 jiL mobile phase, centrifuge at 14000 rpm for 5 min. Add 200 |xL hexane, mix for 15 sec, centrifuge at 14000 rpm for 3 min. Inject a 100 |xL aliquot of the aqueous phase. (Protect sample from light!)
HPLC VARIABLES

Guard column: 4 X 4 5 |xm LiChrospher RP 8 Column: 250 X 4.6 5 (xm LiChrosorb RP 8 Mobile phase: MeOHibuffer 70:30 adjusted to pH 3.0 with orthophosphoric acid (Buffer was 5 mM heptanesulfonic acid containing 50 mM ammonium perchlorate and 1.32% (v/v) triethylamine.) Column temperature: 35 Flow rate: 1.2 Injection volume: 100 Detector: UV 315
CHROMATOGRAM

Retention time: 5.3-5.4 Internal standard: SI 5 (G.D.Searle) (8.5-8.8) Limit of quantitation: 5 ng/mL
KEY WORDS

plasma
REFERENCE Metz,R.; Muth,R; Ferger,M.; Kukes,V.G.; Vergin,H. Sensitive high-performance liquid chromatographic determination of arbidol, a new antiviral compound in human plasma, J.Chromatogr.A, 1998, 810, 63-69.

Arecoline
Molecular formula: C8H13NO2 Molecular weight: 155.20 CAS Registry No.: 63-75-2, 300-08-3 (HBr) Merck Index: 815 SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jmL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 jim Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 207.5 CHROMATOGRAM Retention time: 3.1 KEYWORDS whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163.

Argatroban
Molecular formula: C23H36N6O5S Molecular weight: 508.64 CAS Registry No.: 74863-84-6 Merck Index: 816

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Rainin 300A C-18 Mobile phase: MeOH:water 33:67 containing 10 mM ammonium acetate Flow rate: 2 Detector: UV 254
CHROMATOGRAM

Retention time: 153 (R), 163 (S)

KEYWORDS

chiral
REFERENCE Rawson,T.E.; VanGorp,K.A.; Yang,J.; Kogan,T.P. Separation of 21-(R)- and 21-(S)-argatroban: solubility and activity of the individual diastereoisomers [letter], J.Pharm.ScL, 1993, 82, 672-673.

Artemisinin
Molecular formula: C15H22O5 Molecular weight: 282.34 CAS Registry No.: 63968-64-9 Merck Index: 856

SAMPLE

Matrix: blood Sample preparation: 1 mL Serum + 4 mL n-chlorobutane, vortex for 30 s, centrifuge at 5000 rpm for 15 min, freeze in acetone/dry ice. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |xL MeOH, add 400 yJL 0.2% NaOH, mix, heat at 45 for 30 min, cool, add 50 JJLL 100 mM acetic acid in MeOH, inject a 200 jxL aliquot.
HPLCVARIABLES

Column: 100 mm long 5 |xm LiChrosorb C18 Mobile phase: MeOH: 10 mM pH 4.5 phosphate buffer 45:55 Flow rate: 0.8 Injection volume: 200 Detector: UV (wavelength not specified)
CHROMATOGRAM

Retention time: 10 Limit of detection: 2.5 ng/mL

KEYWORDS

serum; pharmacokinetics; derivatization

REFERENCE Titulaer,H.A.C; Zuidema,J.; Kager,P.A.; Wetsteyn,J.C.F.M.; Lugt,C.B.; Merkus,F.W.H.M. The pharmacokinetics of artemisinin after oral, intramuscular and rectal administration to volunteers, J.Pharm.PharmacoL, 1990, 42, 810-813. SAMPLE

Matrix: blood Sample preparation: Condition a 4 mm diameter Empore C18 SPE membrane with 0.5 mL MeOH and 0.5 mL water, do not allow to dry. Centrifuge 1 mL serum, add to SPE membrane, wash with 300 |xL water, elute with 100 |xL MeCN:water 65:35, inject a 50 (JLL aliquot of the eluate.
HPLC VARIABLES

Column: 150 X 2 5 \xm Ultrasphere ODS Mobile phase: MeCN:water 50:50 Flow rate: 0.3 Injection volume: 50 Detector: Chemiluminescence in a fluorescence detector with no light source emission wavelength 425 nm. The effluent from the column mixed with reagent pumped at 0.5 mL/ min and flowed through a convoluted mixing coil (1.1 mL dead volume) to the detector. (Reagent was 15 jjig/mL luminol and 30 |xg/mL hematin in 100 mM NaOH. Let stand for 30 min before use. Protect from light. Prepare daily.)
CHROMATOGRAM Retention time: 7

Internal standard: dihydroartemisinin (5)

Limit of detection: 10 ng/mL OTHER SUBSTANCES Noninterfering: arteether, artemether

KEYWORDS

serum; post-column reaction; SPE

REFERENCE Green,M.D.; Mount,D.L.; Todd,G.D.; Capomacchia,A.C. Chemiluminescent detection of artemisinin. Novel endoperoxide analysis using luminol without hydrogen peroxide, J.Chromatogr.A, 1995, 695, 237-242. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 250 fiL saturated NaCl solution, vortex for 5 s, add 5 mL isooctane:l-chlorobutane 45:55, vortex for 3 min, centrifuge at 1440 g for 15 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 50 |JLL EtOH:water 50:50, let stand at 4 for 18 h, deoxygenate with a stream of nitrogen at 5 mL/min for 2 min (J. Chromatogr. 1983, 256, 323), inject
a 20 JULL aliquot. HPLC VARIABLES

Column: 250 x 4 5 |xm Lichrosphere 100 CN Mobile phase: MeCN:50 mM acetic acid 15:85 adjusted to pH 5.0 with 1 M NaOH Column temperature: 30 Flow rate: 1.5 Injection volume: 20 Detector: E, Bioanalytical Systems Model 200A, glassy carbon electrode, Ag/AgCl reference electrode, operated in reductive mode under a helium atmosphere CHROMATOGRAM Retention time: 16.1 Internal standard: artemisinin
OTHER SUBSTANCES

Extracted: artemether, dihydroartemisinin

KEYWORDS

plasma; treat glassware with 5% dichlorodimethylsilane; artemisinin is IS

REFERENCE Navaratnam,V.; Mansor,S.M.; Chin,L.K; Mordi,M.N.; Asokan,M.; Nair,N.K. Determination of artemether and dihydroartemisinin in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies, J.Chromatogr.B, 1995, 669, 289-294. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL 100 mg Bond-Elut phenyl SPE cartridge with 1 mL MeOH and 1 mL 1 M acetic acid. Add 1 mL plasma to the SPE cartridge, wash with two 1 mL portions of 1 M acetic acid, wash with 1 mL MeOHrI M acetic acid 20:80, elute with two 1 mL portions of ethyl acetaterbutyl chloride 20:80. Remove any portion of aqueous phase from the eluate and evaporate the organic portion to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL mobile phase, inject a 50-75 JJLL aliquot.
HPLCVARIABLES

Guard column: Symmetry C8 (Waters)

Column: 150 X 3.9 5 |xm Symmetry C8 (Waters) Mobile phase: MeCN:buffer 50:50 (Buffer was 40 mL 1 M acetic acid and 60 mL 1 M sodium acetate per liter, pH 4.8.) Flow rate: 0.7 Injection volume: 50-75 Detector: UV 290 following post-column reaction. The column effluent mixed with 1.2 M KOH MeOH:water 90:10 pumped at 0.3 mL/min and the mixture flowed through a 1 mL reaction coil (Waters) at 69 to the detector.
CHROMATOGRAM Retention time: 13 OTHER SUBSTANCES

Extracted: artesunic acid, a-dihydroartemisiriin, (S-dihydroartemisinin

KEYWORDS

artemisinin is IS; post-column reaction; plasma; SPE

REFERENCE Batty,K.T.; Davis,T.M.; Thu,L.T.; Binh,T.Q.; Anh,T.K; Ilett,K.F. Selective high-performance liquid chromatographic determination of artesunate and a- and p-dihydroartemisinin in patients with falciparum malaria, J.Chromatogr.B, 1996, 677, 345-350. SAMPLE

Matrix: blood, saliva Sample preparation: 1.5 mL Plasma or saliva + 500 |xL 0.9% NaCl + 2.5 mL ethyl acetate, vortex, centrifuge at 2800 rpm for 3 min, repeat extraction twice more. Combine the organic layers and evaporate them to dryness under a stream of air at room temperature, reconstitute the residue in 100 |xL EtOH, add 400 |xL 0.2% NaOH, heat at 50 for 30 min, cool rapidly in water, wash twice with 500 (xL aliquots of ethyl acetate, centrifuge, evaporate traces of ethyl acetate with a stream of air at room temperature, add 40 |xL 2.5 M acetic acid in EtOH, make up to 500 jxL with MeOH:water 20:80, inject a 200 |xL aliquot.
HPLC VARIABLES

Column: 250 x 4 10 jxm LiChrosorb RP-18 Mobile phase: MeOH:water 40:60 containing 10 mM Na2HPO4-NaH2PO4 Column temperature: 35 1 Flow rate: 1.3 Injection volume: 200 Detector: UV 260
CHROMATOGRAM

Retention time: 12 Limit of detection: 2.5 ng/mL

KEYWORDS

derivatization; plasma; pharmacokinetics

REFERENCE Zhao,S. High-performance liquid chromatographic determination of artemisinine (Qinghaosu) in human plasma and saliva, Analyst, 1987, 112, 661-664. SAMPLE Matrix: leaves

Sample preparation: Dry Artemisia leaves at 40 for 24 h, crush, reflux with 100 mL hexane for 15 min, filter, evaporate hexane to dryness under vacuum at 40. Add 25 mL

Next Page
MeCN to the residue, sonicate, filter (0.45 |uim). Add 100 |xL filtrate to 100 |JLL 1 mg/mL acetophenone in MeCN, inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 15 X 3.2 Brownlee C18 Column: 300 X 3.9 10 fjim fjiBondapak C18 Mobile phase: MeCN:buffer 55:45 (Buffer was 8.3 g sodium acetate and 4 mL glacial acetic acid in 1 mL water, pH 5.1.) Flow rate: 0.45 Injection volume: 50 Detector: UV 289 following post-column derivatization with 1 M KOH in MeOH:water 9:1 at 0.2 mL/min. The mixture flowed through a 4.4 X 0.5 mm (sic) knitted PTFE capillary held at 70.
CHROMATOGRAM

Retention time: 16.5 Internal standard: acetophenone (11.5) Limit of detection: 25 ng

KEY WORDS

post-column reaction
REFERENCE ElSohly,H.N.; Croom,E.M.; ElSohly,M.A. Analysis of the antimalarial sesquiterpene artemisinin in Artemisia annua by high-performance liquid chromatography (HPLC) with postcolumn derivatization and ultraviolet detection, Pharm.Res., 1987, 4, 258-260.

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Ascorbic acid
Molecular formula: C6H8O6 Molecular weight: 176.13 CAS Registry No.: 50-81-7 Merck Index: 867

SAMPLE

Matrix: beverages, vegetables Sample preparation: Blend 5 g food with 25 mL 300 mM trichloroacetic acid for 1 min, make up to 50 mL with 300 mM trichloroacetic acid. Dilute 5 g beverage to 50 mL with 300 mM trichloroacetic acid. Filter (paper) these solutions, dilute with 300 mM trichloroacetic acid to a ascorbic acidoncentration of 1-40 jxg/mL. Remove a 3 mL aliquot ,and add it to 400 |xL 4.5 M pH 6.2 sodium acetate buffer, add an ascorbate oxidase spatula (Boehringer Mannheim), heat at 37 for 2 min, mix, heat at 37 for 3 min, remove the spatula (?), add 500 |xL 0.1% o-phenylenediamine (freshly prepared), mix, heat at 37 in the dark for 30 min, inject a 30 JULL aliquot.
HPLC VARIABLES

Guard column: 20 mm long RP-18 (Bischoff) Column: 125 X 4.6 3 jxm ODS-Hypersil Mobile phase: MeOH:80 mM KH2PO4 20:80, pH 7.8 Flow rate: 1 Injection volume: 30 Detector: F ex 365 (filter) em 418 (filter)
CHROMATOGRAM

Retention time: 8 (ascorbic acid), 10 (isoascorbic acid) Limit of detection: 200 ng/g
KEYWORDS

derivatization; protect from light; avocado; brussels sprouts; cabbage; cauliflower; kale; lemon juice; lettuce; orange juice; paprika; parsley; peas
REFERENCE Speek,A-L; Schrijver,J.; Schreuers,W.H.P. Fluorometric determination of total vitamin C and total isovitamin C in foodstuffs and beverages by high-performance liquid chromatography with precolumn derivatization, J.Agric.Food Chem., 1984, 32, 352-355. SAMPLE

Matrix: beverages, vegetables Sample preparation: 500 |iL Juice or homogenized vegetables + 5 mg pyrogallol + 10 mL 100 mM citric acid, vortex under nitrogen for 1 min, add an equal volume of dichloromethane, vortex for 1 min, centrifuge at 4 at 1200 g for 10 min, repeat dichloromethane wash (if necessary to remove excess fat). Filter (0.45 fim) the aqueous layer, pass a 2 mL aliquot through a conditioned Sep-Pak C18 (?) SPE cartridge, inject an aliquot of the eluate.
HPLC VARIABLES

Column: 150 X 5 DA-X8-11 anion-exchange resin (Dionex) Mobile phase: 100 mM pH 3.8 Citrate buffer containing 10 mM NaCl and 5 mM EDTA Flow rate: 0.5 Injection volume: 100 Detector: F ex Corning 7-60 filter em Wratten 2-E following post-column reaction. The column effluent mixed with the oxidizer pumped at 0.5 mL/min and this mixture flowed

through a 32 cm X 0.25 mm ID stainless-steel coil. The effluent from this coil mixed with the reagent pumped at 0.5 mL/min and this mixture flowed through a 45.7 m X 0.25 mm ID stainless-steel coil at 70 then a 1.5 m X 0.25 mm ID stainless-steel coil at 20 to the detector. (Oxidizer was 2.5 mM mercuric chloride or copper sulfate in mobile phase. Reagent was 3.1 mM o-phenylenediamine in mobile phase.) CHROMATOGRAM Retention time: 23 Limit of detection: 20 ng OTHER SUBSTANCES Extracted: dehydroascorbic acid
KEYWORDS

post-column reaction; bean sprouts; beets; broccoli; grape juice; orange juice; potatoes; tomatoes; SPE
REFERENCE Vanderslice,J.T.; Higgs,D.J. HPLC analysis with fluorometric detection of vitamin C in food samples, J.Chromatogr.ScL, 1984, 22, 485-489. SAMPLE

Matrix: blood Sample preparation: Dilute an aliquot of plasma or serum with an equal volume of 10% metaphosphoric acid, add IS to a final concentration of 4.5 jxg/mL, centrifuge at 3300 g for 10 min, filter the supernatant (0.22 |xm), inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 5 jxm Spherisorb ODS C18 Mobile phase: 50 mM pH 4.5 KH2PO4 containing 5 mM cetyltrimethylammonium bromide Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 8.7 Internal standard: 4-hydroxyacetanilide (13.9) Limit of quantitation: 1 jig/mL OTHER SUBSTANCES Simultaneous: dehydroascorbic acid
KEY WORDS

plasma; serum
REFERENCE Esteve,M.J.; Farre,R.; Frigola,A.; Garcia-Cantabella,J.M. Determination of ascorbic and dehydroascorbic acids in blood plasma and serum by liquid chromatography, J.Chromatogr.B, 1997, 688, 345-349. SAMPLE

Matrix: blood Sample preparation: Collect 5 mL whole blood in a tube with 100 |ULL glutathione solution. Remove a 1 mL aliquot and add it to 4 mL 300 mM trichloroacetic acid, vortex thoroughly for 10 min, let stand in the dark at 4 for 10 min, mix, let stand in the dark at 4 for 10 min, centrifuge at 4 at 2000 g for 10 min. Remove a 1.5 mL aliquot of the supernatant and add it to 200 [iL 4.5 M pH 6.2 sodium acetate buffer, add an ascorbate oxidase spatula (Boehringer Mannheim), heat at 37 for 2 min, mix, heat at 37 for 3 min, remove the

spatula, add 250 |xL 0.1% o-phenylenediamine (freshly prepared), mix, heat at 37 in the dark for 30 min, inject a 20 |xL aliquot. (Prepare glutathione solution by dissolving 1.5 g glutathione in 25 mL water, adjust pH to 6.5 with 2 M NaOH, add 2.25 g ethyleneglycolbis-(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), adjust pH to 6.5 with 10 M NaOH.)
HPLCVARIABLES

Column: 80 X 4.6 3 \xm ODS-Hypersil Mobile phase: MeOH:80 mM KH2PO4 20:80, pH 7.8 Flow rate: 1 Injection volume: 20 Detector: F ex 365 (filter) em 418 (filter)
CHROMATOGRAM

Retention time: 3 Limit of detection: 200 nM

KEY WORDS

derivatization; protect from light; whole blood

REFERENCE Speek,A.J.; Schrijver,J.; Schreurs,W.H. Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization, J.Chromatogr., 1984, 305, 53-60. SAMPLE

Matrix: blood Sample preparation: Dilute plasma with an equal volume of cold 10% metaphosphoric acid containing 0.54 mM disodium EDTA, centrifuge, mix the supernatant with an equal volume of cold 5% metaphosphoric acid containing 0.54 mM disodium EDTA and 10 fig/ mL isoascorbic acid, dilute 25 fold with cold 1.04 mM cysteine containing 0.54 mM EDTA, filter (0.2 |xm), inject a 50 (xL aliquot of the filtrate. (Maintain at 4 during sample preparation and in autosampler.)
HPLC VARIABLES

Guard column: 30 x 4.6 5 fjim RP-18 (Brownlee) Column: 250 x 4.6 5 jjim Ultrasphere ODS Mobile phase: MeOH:buffer 7.5:92.5 adjusted to pH 4.75 with glacial acetic acid (Buffer was 40 mM sodium acetate containing 0.54 mM disodium EDTA and 1.5 mM dodecyltrimethylammonium phosphate.) (At the end of each week wash column with 50-100 mL water and 50-100 mL MeOH, store in MeOH.) Flow rate: 0.8 Injection volume: 50 Detector: E, Bioanalytical systems LC4B, glassy carbon working electrode, stainless steel electrode top, Ag/AgCl reference electrode, +0.5 V
CHROMATOGRAM

Retention time: 14.8 Internal standard: isoascorbic acid (16.1) Limit of detection: 0.02 ng Limit of quantitation: 0.2 ng
KEYWORDS

plasma

REFERENCE Kutnink,M.A.; Hawkes,W.C; Schaus,E.E.; Omaye,S.T. An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components, Anal. Biochem., 1987,166, 424-430. SAMPLE

Matrix: blood Sample preparation: 20 |xL Plasma + 10 |xL 125 |xg/inL a-methyl-L-dopa in water, add to a PCPure hydroxyapatite SPE cartridge (Moritex or Koken), elute with buffer, collect 800 IxL eluate, inject a 20 |xL aliquot. (Buffer was freshly prepared 10 mM pH 6.8 sodium phosphate buffer containing 2.5 mg/mL L-cysteine.)
HPLC VARIABLES

Column: 150 X 4.6 5 \xm Inertsil ODS-2 Mobile phase: 100 mM KH2PO4 containing 1 mM disodium EDTA, adjusted to pH 3 with phosphoric acid Flow rate: 0.6 Injection volume: 20 Detector: E, Irica 2875, 300 mV, Ag/AgCl reference electrode
CHROMATOGRAM

Retention time: 3.5 Internal standard: a-methyl-L-dopa (15) Limit of detection: 240 ng/mL
KEY WORDS

plasma; SPE
REFERENCE Iwase,H; Ono,I. Determination of ascorbic acid in human plasma by high-performance liquid chromatography with electrochemical detection using a hydroxyapatite cartridge for precolumn deproteinization, J.Chromatogr.B, 1994, 655, 195-200. SAMPLE Matrix: blood

Sample preparation: Add one volume of 10% metaphosphoric acid to 3 volumes of plasma, freeze the clear supernatant at -80, thaw, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4 4 um triacontyl Daltosil 100 C30 (Serva) Mobile phase: 13.61 g/L KH2PO4 adjusted to pH 2.34 with concentrated orthophosphoric acid Flow rate: 0.5 for 10 min then 1 for 5 min Injection volume: 10 Detector: UV 250 CHROMATOGRAM Retention time: 9.9 Limit of detection: 2 ng
KEYWORDS

plasma
REFERENCE Manoharan,M.; Schwille,P.O. Measurement of ascorbic acid in human plasma and urine by high-performance liquid chromatography. Results in healthy subjects and patients with idiopathic calcium urolithiasis, J.Chromatogr.B, 1994, 654, 134-139.

SAMPLE

Matrix: blood Sample preparation: 600 |xL Plasma + 600 |xL 10% metaphosphoric acid, freeze, store at -80, allow to thaw at 4 for 1 h, centrifuge at 4 at 1000 g for 10 min, inject a 20 fiL aliquot of the supernatant
HPLCVARIABLES

Guard column: 20 X 4 30-40 |xm Perisorb pellicular C18 (Anachem) Column: 250 X 4.6 5 |xm Nucleosil ODS Mobile phase: MeCNrbuffer 7.5:92.5, pH 5.5 (Buffer was 25 mM myristyltrimethylammonium bromide containing 50 mM NaOH, 60 mM acetic acid, 100 jxg/mL homocysteine, and 200 |xg/mL EDTA.) Flow rate: 0.55 Injection volume: 20 Detector: UV 262 CHROMATOGRAM Retention time: 5.32 OTHER SUBSTANCES Extracted: uric acid
KEYWORDS

plasma
REFERENCE Ross,M.A. Determination of ascorbic acid and uric acid in plasma by high-performance liquid chromatography, J.Chromatogr.B, 1994, 657, 197-200. SAMPLE

Matrix: blood Sample preparation: 30 u,L Plasma + 60 \LL stabilizing solution, mix, let stand on ice for 10-15 min, centrifuge at 4 at 12000 g for 5 min, inject a 1-5 |iL aliquot of the supernatant. (Stabilizing solution was MeOH:water 90:10 saturated with EDTA.)
HPLC VARIABLES

Column: 250 X 4.5 QC Pack C18 (IRICA) or 250 X 4.5 TSK gel ODS 120A (Toyo Soda) Mobile phase: MeOH:water 20:80 containing 50 mM sodium phosphate, 50 mM sodium acetate, 189 jxm dodecyltrimethylammonium chloride, 36.6 |xm tetraoctylammonium bromide, 0.2 mM EDTA, pH adjusted to 4.8 with phosphoric acid (Dissolve dodecyltrimethylammonium chloride in MeOH first.)

Flow rate: 1 Injection volume: 1-5

Detector: E, IRICA Sigma 875, +350 mV, Ag/AgCl reference electrode
CHROMATOGRAM

Retention time: 3 Limit of detection: 0.1 ng

KEYWORDS

plasma
REFERENCE UmegakijK.; Inoue,K.; Takeuchi,N.; Higuchi,M. Improved method for the analysis of ascorbic acid in plasma by high-performance liquid chromatography with electrochemical detection, J.Nutr. Sci.VitaminoUTokyo), 1994, 40, 73-79.

SAMPLE

Matrix: blood, food, formula, perfusate Sample preparation: Serum, perfusate. Vortex 1.5 mL plasma or perfusate with 160 |xL 400 mg/mL metaphosphoric acid in water for 15 s, add 400 |xL MeCN vortex for 15 s, centrifuge at 4 at 1000 g for 30 min, inject an aliquot (Clin.Chem. 1988, 34, 2217). Infant formula. Mix 5 g formula with 40 mL 50 mM monobasic potassium phosphate in water containing 1 g/L dithiothreitol, allow to dissolve. Add 10 mL 500 g/L metaphosphoric acid in water and 20 mL MeCN, mix, centrifuge at 1000 g for 15 min at 5. Remove a 4 mL aliquot of the clear lower layer, dilute with 50 mM monobasic potassium phosphate in water containing 1 g/L dithiothreitol, inject an aliquot. Food (for total ascorbic acid). Suspend 1 g pureed food in 5 mL water, vortex for 15 s, add 1 mL 500 mM dibasic potassium phosphate in water containing 100 g/L dithiothreitol, vortex for 15 s, let stand at room temperature for 30 min, add 1 mL 400 g/L metaphosphoric acid in water, add 2 mL MeCN, vortex for 15 s, centrifuge at 1000 g for 30 min at 5, inject an aliquot of the clear lower layer.
HPLC VARIABLES

Column: 250 X 4.6 Capcell Pak NH2 (Shiseido, Japan) Mobile phase: MeCN:bufFer 80:20 (Prepare mobile phase as follows. Dissolve 680 mg monobasic potassium phosphate in 200 mL water, add 800 mL MeCN and 7.5 mL concentrated phosphoric acid.) Column temperature: 40 Flow rate: 1 Detector: E, Model 400 (EG & G, Princeton Applied Research, USA), 700 mV CHROMATOGRAM Retention time: 4.2 OTHER SUBSTANCES Extracted: dithiothreitol, uric acid
KEY WORDS

human; serum; rat

REFERENCE Margolis,S.A.; Schapira,R.M. Liquid chromatographic measurement of L-ascorbic acid and D-ascorbic acid in biological samples, J.Chromatogr.B, 1997, 690, 25-33. SAMPLE

Matrix: blood, formulations Sample preparation: Tablets. Powder tablets, dissolve in water, inject a 10 |xL aliquot. Injections. Dilute with water, inject a 10 |xL aliquot. Plasma. Condition a Lichrolut RP18 (Merck) SPE cartridge with 3 mL MeOH and 3 mL water. 40 jxL Plasma + 80 |ULL MeCN, mix for 2 min, add 100 |xL water, centrifuge at 3500 rpm for 15 min, evaporate the supernatant under nitrogen at 45 to remove the organic solvents, add slowly to the SPE cartridge, collect the eluate. Evaporate to dryness under a stream of nitrogen at 45. Reconstitute the residue with 500 |xL MeOH containing 4.2 |xg/mL IS. Inject a 10 (xL aliquot.
HPLC VARIABLES

Column: 250 X 4 5 jjim Lichrosorb RP-18 Mobile phase: Gradient. A was MeOH. B was 50 mM ammonium acetate. A:B from 5:95 to 15:85 over 6 min, to 30:70 over 7 min, maintain at 30:70 over 7 min Flow rate: 1 Injection volume: 10 Detector: UV 270

CHROMATOGRAM

Retention time: 1.89 Internal standard: xanthine (4.56)

Limit of detection: 2.5 ng
OTHER SUBSTANCES

Extracted: folic acid, niacin, niacinamide, riboflavin, vitamin B12

KEYWORDS

plasma; SPE; tablets; injections

REFERENCE

PapadoyannisJ.N.; Tsioni,G.K.; Samanidou,V.F. Simultaneous determination of nine water and fat soluble vitamins after SPE separation and RP-HPLC analysis in pharmaceutical preparations and biological fluids, J.Liq.Chromatogr.Rel.Technol, 1997, 20, 3203-3231.
SAMPLE

Matrix: blood, tissue Sample preparation: Plasma. 200 |xL Plasma + 200 fxL 10% metaphosphoric acid, mix, centrifuge at 0 at 6000 g for 10 min, filter (Millipore Molcut II), inject a 10 JJLL aliquot of the filtrate. Liver. Homogenize 500 mg liver with 10 mL 5% metaphosphoric acid, mix, centrifuge at 0 at 6000 g for 10 min, filter (Millipore Molcut II), inject a 10 (JLL aliquot of the filtrate.
HPLC VARIABLES

Column: 300 X 7.9 10 jxm Shimpack SCR-101H (Shimadzu) Mobile phase: 5 mM oxalic acid Column temperature: 30 Flow rate: 0.8 Injection volume: 10 Detector: UV 300 following post-column reaction. The column effluent mixed with 100 mM sodium borohydride in 100 mM NaOH pumped at 0.6 mL/min, the mixture flowed through a "j" type reaction coil (Shimadzu) maintained at 30 to the detector.
CHROMATOGRAM

Retention time: 16.8 Limit of detection: 100 ng/mL OTHER SUBSTANCES Extracted: dehydroascorbic acid
KEYWORDS

fish; carp; yellowtail; liver; plasma; post-column reaction

REFERENCE

Ito,T.; Murata,H.; Yasui,Y.; Matsui,M.; Sakai,T.; Yamauchi,K. Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography, J.Chromatogr.B, 1995, 667, 355-357.
SAMPLE

Matrix: blood, urine Sample preparation: Plasma. 1 mL Plasma + 3 mL 10 g/L metaphosphoric acid, mix, centrifuge at 1500 g. Mix a 500 |xL aliquot of the supernatant with 50 u,L IS solution, inject a 20 J U L L aliquot. Urine. Dilute 1 mL urine with 3 mL 10 g/L metaphosphoric acid. Mix a 500 u,L aliquot with 50 J U L L IS solution, inject a 20 fxL aliquot. (IS solution was 100 mg/L D-isoascorbic acid in 10 g/L metaphosphoric acid containing 2 mM EDTA (nitrogensaturated).)

HPLC VARIABLES

Guard column: 70 X 2 10 fxm PRP-I (Hamilton) Column: 75 X 4.6 3 |xm Ultrasphere ODS Mobile phase: 0.15 mM Hexadecyltrimethylammonium bromide containing 0.5 mM sodium acetate and 0.15 mM disodium EDTA, adjusted to pH 4.0 with acetic acid. (Condition system with twenty 20 fiL aliquots of 10 g/L metaphosphoric acid.) Flow rate: 0.5 Injection volume: 20 Detector: F ex 365 em 440 following post-column reaction. The column effluent mixed with the reagent pumped at 1.5 mL/min and flowed through a 20 m X 0.55 mm ID PTFE coil at 65 then a 1.5 m X 0.55 mm ID PTFE coil at 20 to the detector. (Reagent was 2.5 mM cupric sulfate containing 52 mM citric acid and 0.5 mM 4,5-dimethyl-o-phenylenediamine dihydrochloride, adjusted to pH 4.1 with saturated NaOH. Prepare fresh each day. Prepare 4,5-dimethyl-o-phenylenediamine dihydrochloride by dissolving 4,5-dimethyl-o-phenylenediamine in a minimum volume of diethyl ether, pass anhydrous hydrogen chloride through the solution for 20 min, precipitate the salt with diethyl ether. Wash the salt three times with diethyl ether.)
CHROMATOGRAM

Retention time: 15.5 Internal standard: isoascorbic acid (19.9) Limit of detection: 10 ng
OTHER SUBSTANCES

Extracted: dehydroascorbic acid, dehydroisoascorbic acid

KEY WORDS

post-column reaction; plasma

REFERENCE Lopez-Anaya,A.; Mayersohn,M. Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with fluorescence detection, and comparison with a colorimetric assay, Clin.Chem., 1987, 33, 1874-1878. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 uL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) uL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 249.9

CHROMATOGRAM Retention time: 2.928 KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: food Sample preparation: Blend food with 5% metaphosphoric acid containing L-cysteine, centrifuge at 4 at 57600 g for 10 min, filter (0.45 jxm), immediately inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES

Column: 250 X 4.6 C18 (Beckman) Mobile phase: MeOH:water 75:25 Flow rate: 0.5 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 10 Limit of detection: 1.2 jig/mL

KEYWORDS

comparison with capillary electrophoresis; fruit; vegetables

REFERENCE Choi,O.-K.; Jo,J.-S. Determination of L-ascorbic acid in foods by capillary zone electrophoresis, J.ChromatogrA, 1997, 781, 435-443. SAMPLE

Matrix: formulations Sample preparation: Tablets without iron. Grind 5 tablets to a fine powder, add 10 mL monothioglycerol and 800 mL buffer, sonicate for 30 min, add 150 mL MeOH, make up to 1 L with buffer, filter (GF/C paper), discard first few mL, remove a 10 mL aliquot, make up to 25 mL with mobile phase, inject an aliquot. Tablets with dioctyl sodium sulfosuccinate. Grind 5 tablets to a fine powder, add 10 mL 2-monothioglycerol and 1 g barium chloride, make up to 1 L with buffer, stir vigorously for 30 min, filter (GF/C paper), discard first few mL, inject an aliquot. Capsules with iron. Contents of one capsule + 5 mL 2-monothioglycerol + 2 mL glacial acetic acid + 75 mL buffer, sonicate for 5 min, make up to 100 mL with buffer, stir vigorously for 30 min, filter (GF/C paper), add 300 mg cupferron, stir for 10 min, let stand for 1 h at room temperature, filter (GF/C paper), let stand for 30 min, filter again (if necessary), discard first few mL, inject an aliquot. (Buffer was 48 mL glacial acetic acid and 10 mL triethylamine in 1 L water, adjust pH to 3.6 0.05 with acetic acid or triethylamine, make up to 1.7 L with water.)
HPLC VARIABLES

Column: 100 X 8 Radial Pak A C18 (Waters) Mobile phase: MeOH.buffer 15:85 (Buffer was 2.20 g sodium heptanesulfonate, 100 mg EDTA, 48 mL glacial acetic acid, and 10 mL triethylamine made up to 1.7 L with water, adjust pH to 3.6 0.05 with acetic acid or triethylamine.) Flow rate: 2

Injection volume: 10 Detector: UV 254

CHROMATOGRAM Retention time: 1.3 OTHER SUBSTANCES

Simultaneous: niacinamide (UV 280), thiamine (UV 280), riboflavin (UV 280), pyridoxine (UV 280)
KEY WORDS

multi-vitamin; protect from light; tablets; capsules

REFERENCE Lam,F.-L.; HolcombJ.J.; Fusari,S.A. Liquid chromatographic assay of ascorbic acid, niacinamide, pyridoxine, thiamine, and riboflavin in multivitamin-mineral preparations, J.Assoc.Off.Anal.Chem., 1984, 67, 1007-1011. SAMPLE

Matrix: formulations Sample preparation: Pulverize tablets and weigh out 1 g, add 1 mL formic acid, add 25 mL MeOH, shake mechanically for 10 min, make up to 50 mL with methanol. Remove 10 mL and centrifuge. 5 mL Supernatant + 5 mL 0.0025% p-hydroxybenzoic acid in MeOHrwater 20:80, make up to 25 mL with water, inject an aliquot. (Analyze within 1 h.)
HPLC VARIABLES

Column: 250 X 4.6 LiChrosorb RP8 Mobile phase: MeOH:200 mM pH 3.5 phosphate buffer:water 20:10:70 Flow rate: 1 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 3 Internal standard: p-hydroxybenzoic acid (18)

OTHER SUBSTANCES

Simultaneous: aspirin, p-aminophenol, 3-O-acetylascorbic acid, 2-O-acetylascorbic acid, saccharin, acetaminophen, O-acetyl-p-aminophenol, salicylic acid (UV 280), diacetyl-paminophenol (UV 280)
KEYWORDS

tablets
REFERENCE Thomis,R.; Roets,E.; Hoogmartens,J. Analysis of tablets containing aspirin, acetaminophen, and ascorbic acid by high-performance liquid chromatography, J.Pharm.Sci., 1984, 73, 1830-1833. SAMPLE

Matrix: formulations Sample preparation: Dilute injections with water, inject a 50 |xL aliquot. Dissolve tablets or capsule contents in water (warm if necessary), filter (0.5 fim PTFE), inject a 50 U | LL aliquot of the filtrate. (Dissolve tablets or other formulations containing proteinaceous material in water at 60, add 5% trichloroacetic acid (to pH 4.4), filter, inject a 50 |JLL aliquot.)

HPLC VARIABLES

Guard column: pellicular Corasil Column: 10 urn ixBondapak C18 Mobile phase: Gradient. A was prepared by dissolving 1 g sodium dioctylsulfosuccinate in 170 mL MeOH, add 10 mL concentrated formic acid, make up to 800 mL with water, adjust pH to 2.5 with 1 M KOH, make up to 1 L. B was prepared by dissolving 1 g sodium dioctylsulfosuccinate in 450 mL MeOH, add 10 mL concentrated formic acid, make up to 800 mL with water, adjust pH to 4.6, make up to 1 L. A:B from 100:0 to 0:100 over 25 min (concave curve 9), maintain at 0:100 for 3 min, return to initial conditions over 2 min. Flow rate: 1.5 Injection volume: 50 Detector: UV 280
CHROMATOGRAM Retention time: 2 OTHER SUBSTANCES

Simultaneous: folic acid, niacin (UV 254), niacinamide (UV 254), pyridoxamine, thiamine (UV 254), riboflavin (UV 254), pyridoxine
KEYWORDS

injections; capsules; tablets

REFERENCE Woollard,D.C. New ion-pair reagent for the high-performance liquid chromatographic separation of Bgroup vitamins in Pharmaceuticals, J.Chromatogr., 1984, 301, 470-476. SAMPLE

Matrix: formulations Sample preparation: Weigh out 500 mg ground tablets, extract with water, make up to 50 or 100 mL with water, filter, inject an aliquot.
HPLC VARIABLES

Column: 250 x 4.6 Nucleosil 10 C18 Mobile phase: MeOH:l% acetic acid 25:75 Flow rate: 1 Injection volume: 20 Detector: UV 270
CHROMATOGRAM Retention time: 2.8 OTHER SUBSTANCES

Simultaneous: menadione hydrogen sulfite, niacinamide, riboflavin, thiamine Interfering: pyridoxine

KEYWORDS

tablets; multi-vitamin
REFERENCE Sadlej-Sosnowska,N.; Blitek,D.; Wilczynska-Wojtulewicz,I. Determination of menadione sodium hydrogen sulphite and nicotinamide in multivitamin formulations by high-performance liquid chromatography, J.Chromatogr., 1986, 357, 227-232. SAMPLE

Matrix: formulations

HPLC VARIABLES Column: 100 X 4 3 ^m Hypersil BDS-C18 Mobile phase: Gradient. MeCN:water adjusted to pH 2.1 from 0.3:99.7 to 25:75 over 11 min Flow rate: 0.5 Detector: UV 220 CHROMATOGRAM Retention time: 2.3 OTHER SUBSTANCES Simultaneous: biotin, caffeine, citric acid, folic acid, niacinamide, niacin, pantothenic acid, riboflavin, saccharin, thiamine, pyridoxine, vitamin B12 KEYWORDS tablets REFERENCE Hewlett Packard Leaflet 12-5091-7351 EUS, 1993, 1993, SAMPLE Matrix: fruit, vegetables Sample preparation: Juices. Dilute 2 mL fruit juice to 50 mL with 0.05% disodium EDTA in 100 mM sulfuric acid, filter (Whatman No. 1 paper), inject a 10 |xL aliquot of the filtrate. Fruit, vegetables. Blend (Waring) fruit or vegetable with 0.05% disodium EDTA in 100 mM sulfuric acid for 3 min, centrifuge at 15000 rpm for 10 min. Remove the supernatant and make it up to 10 mL with 0.05% disodium EDTA in 100 mM sulfuric acid, inject a 10 jxL aliquot. HPLC VARIABLES Guard column: MicroGuard ion exclusion cartridge (Bio-Rad) Column: 300 X 7.8 Aminex HPX-87 (Bio-Rad) Mobile phase: 4.5 mM Sulfuric acid Flow rate: 0.5 Injection volume: 10 Detector: UV 245 CHROMATOGRAM Retention time: 12.9 Limit of quantitation: 50 ng KEYWORDS juice; orange; lemon; grapefruit; pineapple; tomatoes; peas; potatoes; strawberries; green peppers REFERENCE Ashoor,S.H.; Monte,W.C.; Welty,J. Liquid chromatographic determination of ascorbic acid in foods, JAssoc.Off.Anal.Chem., 1984, 67, 78-80. SAMPLE Matrix: juice Sample preparation: 15 g Orange juice + 5 mL 12.5% trichloroacetic acid, centrifuge, filter, inject a 10 jxL aliquot of the filtrate. (To determine ascorbic acid and dehydroascorbic acid (by reducing dehydroascorbic acid to ascorbic acid) dilute filtrate with water to an ascorbic acid concentration of 10-100 (xg/mL adjust pH to 7.0. Remove a 500 |xL aliquot and add it to 2 mL 0.8% DL-homocysteine, let stand for 15 min, filter, inject a 10 |xL aliquot.)

HPLC VARIABLES

Column: 250 X 4.6 5 fjim Erbasil NH2 Mobile phase: MeOH:0.25% pH 3.5 KH2PO4 50:50 Flow rate: 0.5 Injection volume: 10 Detector: UV 254
CHROMATOGRAM Retention time: 8.7 KEYWORDS

orange juice; comparison with capillary electrophoresis

REFERENCE Chiari,M.; Nesi,M.; Carrea,G.; Righetti,P.G. Determination of total vitamin C in fruits by capillary zone electrophoresis, J.Chromatogr., 1993, 645, 197-200. SAMPLE

Matrix: juice Sample preparation: Dilute with water to a ascorbic acid concentration of 10 |xg/mL, filter (0.45 fjim), remove a 20 jxL aliquot of the nitrate and add it to 10 jxL 125 jxg/mL a-methylL-dopa in water and 800 |xL 2% metaphosphoric acid, inject a 20 JJLL aliquot. (To measure total ascorbic acid content after reduction of dehydroascorbic acid dilute juice with water to a ascorbic acid concentration of 10 |ig/mL, filter (0.45 |xm), remove a 20 |xL aliquot of the filtrate and add it to 10 |xL 125 |xg/mL a-methyl-L-dopa in water and 800 |xL buffer, let stand for 15 min, inject a 20 |xL aliquot. (Buffer was freshly prepared 10 mM pH 6.8 sodium phosphate buffer containing 2.5 mg/mL L-cysteine.))
HPLC VARIABLES

Column: 150 X 4.6 5 n-m Inertsil ODS-2 Mobile phase: 100 mM KH2PO4 containing 1 mM disodium EDTA, adjusted to pH 3 with phosphoric acid Flow rate: 0.6 Injection volume: 20 Detector: E, Irica 875, 300 mV, Ag/AgCl reference electrode
CHROMATOGRAM

Retention time: 3.5 Internal standard: a-methyl-L-dopa (15) Limit of detection: 0.15 ng
KEYWORDS

lemon; grape; orange; apple; tea

REFERENCE Iwase,H.; Ono,I. Determination of ascorbic acid and dehydroascorbic acid in juices by high-performance liquid chromatography with electrochemical detection using L-cysteine as precolumn reductant, J.ChromatogrA, 1993, 654, 215-220. SAMPLE

Matrix: plants Sample preparation: Pulverize 200 mg plant tissue in liquid nitrogen, extract twice with a total volume of 3% metaphosphoric acid containing 1 mM EDTA, centrifuge, pass through a conditioned C18 SPE cartridge (Millipore), inject an aliquot of the last 500 |xL oftheeluate.;SPE
HPLC VARIABLES

Column: 250 X 4.6 3 |xm C18 (Bio-Rad)

Mobile phase: pH 2.5 Phosphoric acid containing 0.1 mM EDTA Flow rate: 0.8 Injection volume: 20 Detector: UV 248 CHROMATOGRAM Retention time: 3.2
KEYWORDS comparison with capillary electrophoresis; SPE REFERENCE Davey,M.W.; Bauw,G.; Montagu,M.V. Analysis of ascorbate in plant tissues by high-performance capillary zone electrophoresis, Anal.Biochem., 1996, 239, 8-19.

SAMPLE Matrix: solutions

HPLC VARIABLES

Column: 250 X 4.6 Capcell Pak NH2 (Shiseido, Japan) Mobile phase: MeCN:buffer 80:20 (Prepare mobile phase as follows. Mix 680 mg monobasic potassium phosphate, 7.5 mL concentrated phosphoric acid with 200 mL water, add 800 mL MeCN.) Column temperature: 40 Flow rate: 1.0 Detector: E, Model 400 (EG & G, Princeton Applied Research, Princeton, NJ), 700 mV CHROMATOGRAM Retention time: 4.4
OTHER SUBSTANCES

Simultaneous: dithiothreitol, isoascorbic acid, metaphosphoric acid, uric acid,

REFERENCE Margolis,S.A.; Duewer,D.L. Measurement of ascorbic acid in human plasma and serum: stability, intralaboratory repeatability, and interlaboratory reproducibility, Clin.Chem., 1996, 42, 1257-1262. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject
a 10 JJLL aliquot. HPLC VARIABLES

Column: 300 X 3.9 10 |xm jjiBondapak C18 Mobile phase: MeOHracetic acid:triethylamine:water 40:1.5:0.5:58 Flow rate: 1.5 Injection volume: 10 Detector: UV 261 CHROMATOGRAM Retention time: 3
OTHER SUBSTANCES

Simultaneous: salicylic acid, benzoic acid, quinine, dihydroquinine

REFERENCE Roos,R-W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE Matrix: solutions HPLC VARIABLES Column: 33 x 4.6 3 |xm Supelcosil LC-8-DB Mobile phase: MeOHrbuffer 15:85 (Buffer was 4.3 mM sodium hexanesulfonate containing 0.1% triethylamine, adjusted to pH 2.8 with phosphoric acid.) Column temperature: 35 Flow rate: 1 Detector: UV 200 CHROMATOGRAM Retention time: 0.5 OTHER SUBSTANCES Simultaneous: niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, niacinamide REFERENCE Rainin Catalog, Cl-94, 1994, p. 780. SAMPLE Matrix: solutions HPLC VARIABLES Column: 100 X 4.6 Spheri-5 RP-8 Mobile phase: Gradient. A was 100 mM pH 4.7 acetate buffer. B was MeCN: 100 mM pH 4.7 acetate buffer 25:75. Column temperature: 26 Flow rate: 4 Detector: UV 254 CHROMATOGRAM Retention time: 0.6 OTHER SUBSTANCES Simultaneous: niacin, niacinamide, pyridoxine, riboflavin, thiamine REFERENCE Rainin Catalog, Cl-94, 1994, p. 7.21. SAMPLE Matrix: tissue Sample preparation: Deproteinize heart tissue with ice-cold 600 mM perchloric acid, neutralize, centrifuge at 4 at 26890 g for 15 min, filter (0.45 (xm) the supernatant, inject a 20 |xL aliquot of the filtrate. HPLC VARIABLES Guard column: 20 x 4.6 3 |xm LC-18-T (Supelco) Column: 150 X 4.6 3 ^m LC-18-T (Supelco) Mobile phase: Gradient. A was MeOH:10 mM KH 2 PO 4 1:99, pH 7.0. B was MeOH:100 mM KH2PO4 containing 2.8 mM tetrabutylammonium hydroxide 30:70, pH 5.5. A:B 100:0 for

12 min, to 60:40 over 2 min, to 56:44 over 11 min, to 0:100 over 10 min, maintain at 0: 100 for 5 min, re-equilibrate at initial conditions for 5 min. Flow rate: 1.2 Injection volume: 20 Detector: UV 266
CHROMATOGRAM Retention time: 6 OTHER SUBSTANCES

Extracted: adenosine, adenosine triphosphate

KEYWORDS

rat; heart
REFERENCE Lazzarino,G.; Di Pierro,D.; Tavazzi,B.; Cerroni,L.; Giardina,B. Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing highperformance liquid chromatography, Anal.Biochem., 1991, 197, 191-196. SAMPLE

Matrix: urine Sample preparation: Prepare a column by suspending 200-400 mesh Dowex 50W-X8 resin in water and pouring it into a 100 x 7 column, allow to settle, wash with 10 mL 2 M HCl, wash with water until the washings are neutral. Mix urine with an equal volume of 5% metaphosphoric acid containing 0.5% p-thiodiglycol. Add a 1 mL aliquot to the column, wash with 3.95 mL 2 mM tartaric acid containing 0.05% p-thiodiglycol, collect all the effluent from the column in a tube containing 50 |xL 5% disodium EDTA cooled in ice, filter (0.45 |xm) the eluate, inject a 50-250 |xL aliquot.
HPLC VARIABLES

Column: two 50 X 7.6 Asahipak GS-320 hydrophilic gel columns in series Mobile phase: 2.25 g/L Tartaric acid containing 0.75 g/L disodium EDTA and 0.5 g/L pthiodiglycol, adjusted to pH 3.00-3.03 with 4 M NaOH Column temperature: 30 Flow rate: 1 Injection volume: 50-250 Detector: F ex 325 em 400 following post-column reaction. The column effluent mixed with 20 mM benzamidine hydrochloride pumped at 0.36 mL/min and with 750 mM pH 10 potassium borate buffer containing 200 mM potassium sulfite pumped at 0.36 mL/min and the mixture flowed through a 50 m X 0.5 mm ID PTFE tube at 90 to the detector.
CHROMATOGRAM Retention time: 48 OTHER SUBSTANCES

Extracted: isoascorbic acid Simultaneous: dehydroascorbic acid, diketogluconic acid, diketogulonic acid
KEY WORDS

post-column reaction; SPE

REFERENCE Seki,T.; Yamaguchi,Y; Noguchi,K; Yanagihara,Y. Determination of ascorbic acid in human urine by high-performance liquid chromatography coupled with fluorimetry after post-column derivatization with benzamidine, J.Chromatogr., 1987, 385, 287-291.

SAMPLE

Matrix: urine Sample preparation: Filter (paper), dilute 6 times with 0.05% metaphosphoric acid, inject an aliquot. Alternatively, dilute with an equal volume of 200 mM dithiothreitol, let stand at room temperature for 30 min, dilute three fold with 0.05% metaphosphoric acid, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4 5 |xm Ultrasphere Mobile phase: 13.61 g/L KH2PO4 adjusted to pH 2.34 with concentrated orthophosphoric acid Flow rate: 0.5 for 10 min then 1 for 5 min Injection volume: 10 Detector: UV 250
CHROMATOGRAM

Retention time: 8 Limit of detection: 1.5 ng

REFERENCE Manoharan,M.; Schwille,P.O. Measurement of ascorbic acid in human plasma and urine by high-performance liquid chromatography. Results in healthy subjects and patients with idiopathic calcium urolithiasis, J.Chromatogr.B, 1994, 654, 134-139. SAMPLE

Matrix: wine Sample preparation: Adjust pH of wine to 7-8 with potassium bicarbonate. Remove a 1 mL aliquot and add it to 1 mL 170 mM phenacyl bromide in acetone, add 1 mL 17 mM 18-crown-6 in acetone, add 1 mL acetone, heat in a boiling water bath for 75 min, cool, inject a 10 JJLL aliquot. (Recrystallize phenacyl bromide from n-heptane.)
HPLC VARIABLES

Guard column: 37-50 |xm Bondapak C18/Corasil Column: 250 X 4 7 jxm RP-18 (Merck) Mobile phase: Gradient. MeOH:water from 35:65 to 85:15 over 20 min. Flow rate: 2 Injection volume: 10 Detector: UV 254
CHROMATOGRAM Retention time: 11.1 OTHER SUBSTANCES

Extracted: acetic acid, anisic acid, benzilic acid, benzoic acid, butyric acid, caprylic acid, cinnamic acid, citramalic acid, citric acid, enanthic acid, fumaric acid, galacturonic acid, gallic acid, glutaric acid, glycolic acid, glyoxylic acid, p-hydroxybenzoic acid, isocitric acid, a-ketoglutaric acid, lactic acid, malic acid, mandelic acid, phenylacetic acid, propionic acid, protocatechuic acid, pyruvic acid, salicylic acid, sorbic acid, succinic acid, tartaric acid, valeric acid, vanillic acid
KEYWORDS

derivatization
REFERENCE Mentasti,E.; Gennaro,M.C; Sarzanini,C; Baiocchi,C; Savigliano,M. Derivatization, identification and separation of carboxylic acids in wines and beverages by high-performance liquid chromatography, J.Chromatogr., 1985, 322, 177-189.

Aspartame
Molecular formula: C14H18N2O5 Molecular weight: 294.31 CAS Registry No.: 22839-47-0 Merck Index: 874

SAMPLE

Matrix: beverages Sample preparation: Filter sample.

HPLC VARIABLES

Column: 150 X 4.5 5 Jim Hiasil C18 (Higgins) Mobile phase: MeOH:25 mM phosphate buffer 45:55, pH 3.0 Flow rate: 1.0 Injection volume: 20 Detector: UV 218
CHROMATOGRAM

Retention time: 3.5 Limit of detection: 1.4 mg/mL OTHER SUBSTANCES Extracted: benzoic acid, caffeine
KEYWORDS

comparison with UV spectrophotometry and capillary electrophoresis; soft drinks

REFERENCE McDevitt,V.L.; Rodriguez,A.; Williams,K.R. Analysis of soft drinks: UV spectrophotometry, liquid chromatography, and capillary electrophoresis, J.Chem.Educ, 1998, 75, 625-629. SAMPLE

Matrix: beverages, formulations Sample preparation: Dilute beverages and formulations with water, inject an aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Spherisorb Hexyl Mobile phase: MeOH:0.1% perchloric acid 15:85, pH 2.8 Flow rate: 1 Detector: E, ESA Coulochem Model 5100A, first electrode (screen mode) +0.10 V, second electrode (measuring electrode) +0.80 V, following post-column reaction. The column effluent flowed through a 20 m X 0.3 mm ID PTFE coil irradiated with a UV 254 lamp to the detector. CHROMATOGRAM Retention time: 13 Limit of detection: 500 ng/mL Limit of quantitation: 1 fxg/mL OTHER SUBSTANCES Simultaneous: caffeine
KEY WORDS

post-column reaction; soft drinks; post-column photochemical derivatization

REFERENCE Galletti,G.C; Bocchini,P. High-performance liquid chromatography with electrochemical detection of aspartame with a post-column photochemical reactor, J.Chromatogr.A, 1996, 729, 393-398. SAMPLE

Matrix: beverages, sweetener Sample preparation: Sweetener. Dissolve 30 mg powdered tabletop sweetener in water and dilute to 25 mL, filter (0.2 jjim PTFE). Beverages. Dilute fruit juice 1:10 with water. Degas carbonated beverages in a ultrasonic bath for 5 min, dilute 1:10 with water, filter. Inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 50 X 4 Dionex IonPak AG4A-SC Column: 250 X 4 Dionex IonPak AS4A-SC Mobile phase: Gradient. A was 1 mM sodium carbonate. B was 12.5 mM sodium carbonate. A:B 100:0 for 4.5 min, from 100:0 to 0:100 over 1 min, maintain at 0:100 for 22.5 min, from 0:100 to 100:0 over 0.1 min Flow rate: 1 Injection volume: 50 Detector: UV 190 for 6 min, UV 206 22 min, then UV 190; Conductivity, Dionex ED40 in conductivity mode preceded by a Dionex ASRS-I suppressor (external water mode, 300 mA)
CHROMATOGRAM

Retention time: 2.5 Limit of detection: 35 ng/mL (UV) OTHER SUBSTANCES Simultaneous: acesulfame, saccharin
REFERENCE Chen,Q.-C; Mou,S.-F.; Liu,K.-R.; Yang,Z.-Y; Ni,Z.-M. Separation and determination of four artificial sweeteners and citric acid by high-performance liquid chromatography, J.Chromatogr.A, 1997, 771, 135-143. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30

Detector: UV 200.5
CHROMATOGRAM Retention time: 9.8 KEYWORDS whole blood REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Aspirin
Molecular formula: C9H8O4 Molecular weight: 180.16 CAS Registry No.: 50-78-2 Merck Index: 886 SAMPLE

Matrix: blood Sample preparation: Add 250 fxL 200 mM orthophosphoric acid to 250 \xL chilled plasma within 10 min of centrifuging (if fresh plasma) or within 10 min of thawing (if frozen plasma), vortex for 20 s, centrifuge at 5800 g for 3 min. Inject a 200 jxL aliquot onto column A and elute to waste with mobile phase A, after 2 min backflush the contends of column A onto column B with mobile phase B, elute with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES

Column: A 10 X 4.3 30 jjim Hypersil C18 PEEK cartridge (Shandon, England); B 10 X 4 30 fxm Hypersil C8 + 250 X 4.6 5 |xm Nucleosil C8 Mobile phase: A Water:orthophosphoric acid 1000:1, pH 2.5; B MeCN:MeOH:water:orthophosphoric acid 150:200:650:1 (pH 2.6) Flow rate: 1 Injection volume: 200 Detector: UV 225
CHROMATOGRAM

Retention time: 33 Limit of detection: 40 ng/mL Limit of quantitation: 100 ng/mL

OTHER SUBSTANCES

Extracted: salicylic acid Noninterfering: barbital, butobarbital, caffeine, 8-chlorotheophylline, clonazepam, cocaine, diazepam, flurazepam, furosemide, hydralazine, imipramine, nitrazepam, phenytoin, pindolol, propranolol, quinidine, theophylline Interfering: xylazine, prazosin
KEYWORDS

column-switching; plasma
REFERENCE McMahon,G.R; Kelly,M.T. Determination of aspirin and salicylic acid in human plasma by columnswitching liquid chromatography using on-line solid-phase extraction, Anal.Chem., 1998, 70, 409414. SAMPLE

Matrix: formulation Sample preparation: Weigh 500 mg homogenized analgesic powder, transfer to 100 mL volumetric flask, add ca. 50 mL mobile phase, swirl and dilute to volume with mobile phase. Dilute an aliquot of this solution 1:10 with mobile phase, filter (0.20 jim Cameo nylon filter, MSI, Westboro, MA) an aliquot, inject an aliquot of the filtrate.
HPLCVARIABLES

Column: 100 X 2.1 5 |xm Hypersil ODS

Mobile phase: MeCN:triethylamine:acetic acidiwater 5.5:0.2:0.2:94.1 (Prepare mobile phase as follows. Mix 110 mL MeCN, 4 mL triethylamine, 4 mL glacial acetic acid and make up to 2 L with water.) Flow rate: 1.5 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 1.4
OTHER SUBSTANCES

Extracted: acetaminophen, caffeine Interfering: salicylic acid

KEYWORDS

powder
REFERENCE Ferguson,G.K. Quantitative HPLC analysis of an analgesic/caffeine formulation: Determination of caffeine, J.Chem.Educ, 1998, 75, 467-469. SAMPLE

Matrix: formulations Sample preparation: Add 50 mL of mobile phase to 0.5 g of sample and swirl to aid dissolution. Dilute to 100 mL with mobile phase. Dilute 1:10, filter (0.22 |xm nylon). Inject a 10 |JLL aliquot.
HPLC VARIABLES

Column: 100 X 2.1 5 |xm Hypersil ODS Mobile phase: MeCN:water:triethylamine:acetic acid 5.5:94.1:0.2:0.2 Flow rate: 1.5 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 1.4
OTHER SUBSTANCES

Simultaneous: acetaminophen, caffeine

KEY WORDS

powder
REFERENCE Ferguson,G.K. Quantitative HPLC analysis of an analgesic/caffeine formulation: Determination of caffeine, J.Chem.Educ., 1998, 75, 467-469. SAMPLE

Matrix: formulations Sample preparation: Sonicate 75 mg powdered tablets with 25 mL mobile phase for 15 min, filter (paper), inject a 135 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrabase C18 Scharlau Science, Spain Mobile phase: MeOH:20 mM pH 4.KH2PO4 30:70 (pH adjusted with orthophosphoric acid) Flow rate: 1.5 Injection volume: 135

Detector: UV 224
CHROMATOGRAM

Retention time: 12.8 Limit of quantitation: 3.6 fJig/mL

OTHER SUBSTANCES

Simultaneous: caffeine, salicylic acid, thiamine

KEYWORDS

tablets
REFERENCE Gamiz-Gracia,L.; Luque de Castro,M.D. An HPLC method for the determination of vitamin Bl, caffeine, acetylsalicylic acid, and the impurities of salicylic acid in a pharmaceutical preparation, J.Liq.Chromatogr.Rel.Technol, 1997, 20, 2123-2133. SAMPLE

Matrix: formulations Sample preparation: Weigh out powdered sample containing 68 mg aspirin, add 80 mL MeOH, sonicate for 10 min, dilute to 100 mL with MeOH, centrifuge. Remove a 5 mL aliquot of the supernatant and add it to 1 mL 2 mg/mL resorcinol, add 2 mL MeOH, make up to 20 mL with 50 mM pH 3.0 triethylamine phosphate, inject an aliquot.
HPLC VARIABLES

Column: 150 X 3.2 5 |xm Hypersil ODS Mobile phase: THF:50 mM pH 3.0 triethylamine phosphate 12:88 Flow rate: 0.6 Injection volume: 20 Detector: UV 275 following post-column reaction. The column effluent flowed through a 10 m X 0.3 mm ID crocheted PTFE coil irradiated with an 8 W low-pressure mercury lamp at 254 nm to the detector.
CHROMATOGRAM

Retention time: 15 Internal standard: resorcinol (9)

OTHER SUBSTANCES

Simultaneous: acetaminophen (post-column irradiation gives little increase in peak height), caffeine (post-column irradiation gives little increase in peak height), propyphenazone (post-column irradiation gives a decrease in peak height)
KEYWORDS

post-column reaction; post-column photochemical derivatization

REFERENCE Di Pietra,A.M.; Gatti,R.; Andrisano,V.; Cavrini,V. Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics, J.ChromatogrA, 1996, 729, 355-361.

Aspoxicillin
Molecular formula: C21H27N5O7S Molecular weight: 493.54 CAS Registry No.: 63358-49-6 Merck Index: 887

SAMPLE

Matrix: blood, tissue, urine Sample preparation: Serum, plasma. Dilute serum or plasma 1:2 to 1:10 with buffer, centrifuge, inject a 20 jxL aliquot of supernatant. Urine. Dilute urine 1:10 to 1:100 with buffer, centrifuge, inject a 20 |xL aliquot of supernatant. Tissue (lung, gut). Cut tissue with a scalpel, homogenize with 1-3 mL buffer, centrifuge at 9600 g for 5 min three times, inject a 20 |ULL aliquot. Tissue (chondral). Cut tissue with a scalpel, homogenize with 3-6 mL buffer in an ice bath for 2-3 min, centrifuge at 9600 g for 5 min four or five times, inject a 100 |xL aliquot. Dilute human pleural samples with buffer, centrifuge, inject a 20 jxL aliquot. (Buffer was 66.6 mM K2HPO4 adjusted to pH 7.40 with KH2PO4.)
HPLC VARIABLES

Column: 200 X 4 5 |xm Nucleosil C18 Mobile phase: MeOH:buffer 8:92, adjusted to pH 5.8 with phosphoric acid (Buffer was 57.4 mM K2HPO4 adjusted to pH 5.8 with phosphoric acid.) Flow rate: 1 Injection volume: 20-100 Detector: UV 220
CHROMATOGRAM

Retention time: 12 Limit of detection: 500 ng/mL

KEYWORDS

serum; plasma; lung; gut; pleural; chondral

REFERENCE Knoller,J.; Konig,W.; Schonfeld,W.; Bremm,K.D.; Koller,M. Application of high-performance liquid chromatography of some antibiotics in clinical microbiology, J.Chromatogr., 1988, 427, 257-267. SAMPLE

Matrix: broncho-alveolar lavage fluid Sample preparation: 1 mL Broncho-alveolar lavage fluid + 100 |xL 5 |xg/mL amoxicillin, vortex for 10 s, filter (Tosoh Ultracent-30 with a molecular mass cut-off at 30000) while centrifuging at 1500 g at 5 for 30 min, inject a 100 |xL aliquot of the ultrafiltrate.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Shodex C18 5A (Showa Denko) Mobile phase: MeCN:50 mM pH 3.0 potassium hydrogen phosphate containing 20 mM sodium 1-heptanesulfonate and 5 mg/L EDTA 10:100 Column temperature: 40 Flow rate: 1.2 Injection volume: 100 Detector: E, Irica X875, glassy carbon electrode 800 mV, Ag/AgCl reference electrode, following 10 m X 0.3 mm PTFE tubing irradiated by a GL-10 10 W mercury lamp

CHROMATOGRAM

Retention time: 24 Internal standard: amoxicillin (17) Limit of detection: 1 ng/mL

KEY WORDS

ultrafiltrate
REFERENCE Yamazaki,T.; Ishikawa,T.; Nakai,H.; Miyai,M.; Tsubota,T.; Asano,K. Determination of aspoxicillin in broncho-alveolar lavage fluid by high-performance liquid chromatography with photolysis and electrochemical detection, J.Chromatogr., 1993, 615, 180-185.

Astemizole
Molecular formula: C28H31FN4O Molecular weight: 458.58 CAS Registry No.: 68844-77-9 Merck Index: 891 Led nicer No.: 3 177

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 [iL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 13.16 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Atenolol
Molecular formula: C14H22N2O3 Molecular weight: 266.34 CAS Registry No.: 29122-68-7 Merck Index: 892 Lednicer No.: 2 109

SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 250 ng albuterol, mix for 10 s, add 10 mL dichloromethane:2-propanol 75:25, shake for 10 min. Centrifuge at 2000 g for 10 min at 4. Remove the organic phase and evaporate it to dryness under a stream of nitrogen at 60. Reconstitute the residue in 200 |xL mobile phase , mix for 10 s. Centrifuge at 6500 g for 10 min. Inject a 40 JULL aliquot of the supernatant.
HPLCVARIABLES

Guard column: 4 X 4 5 fjtm LiChrospher 100 RP-18 Column: 250 x 4 5 \x,m Supelcosil LC-18 (Supelco) Mobile phase: n-Propanol'.buffer 5:95 (Buffer was 50 mM sodium dodecyl sulfate in 10 mM pH 5.8 sodium phosphate buffer.) Flow rate: 1.3 Injection volume: 40 Detector: F ex 222 em 300
CHROMATOGRAM

Retention time: 15.7 Internal standard: albuterol (20.6) Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES

Noninterfering: chlorthalidone, xipamide

KEYWORDS

plasma; pharmacokinetics
REFERENCE Giachetti,C; Tenconi,A.; Canali,S.; Zanolo,G. Simultaneous determination of atenolol and chlorthalidone in plasma by high-performance liquid chromatography. Application to pharmacokinetic studies in man, J.Chromatogr.B, 1997, 698, 187-194. SAMPLE

Matrix: blood Sample preparation: Centrifuge plasma or serum at 11 300 g for 7 min, inject a 200 |xL aliquot onto column A, elute to waste with mobile phase A, after 6 min backflush the contents of column A onto column B with mobile phase B, after 6 min remove column A from the circuit, elute column B with mobile phase B, monitor the effluent from column B. (Reequilibrate column A with mobile phase A for 4 min.)
HPLC VARIABLES

Column: A 20 X 4.0 BioTrap 500 C18 (ChromTech); B 12.5 X 4.6 5 |xm Zorbax SB-CN + 150 x 4.6 5 jjim Zorbax SB-CN Mobile phase: A 2-Propanol:30 mM pH 7.0 sodium phosphate buffer containing 5 mM sodium octanesulfonic acid 2:98; B MeCN:30 mM pH 3.0 sodium phosphate buffer containing 2 mM sodium octanesulfonic acid 25:75

Flow rate: A 0.8; B l Injection volume: 200 Detector: F ex 230 em 300

CHROMATOGRAM Retention time: 9.5 KEYWORDS

plasma; serum; column-switching

REFERENCE Hermansson,J.; Grahn,A.; Hermansson,I. Direct injection of large volumes of plasma/serum on a new biocompatible extraction column for the determination of atenolol, propranolol and ibuprofen. Mechanisms for the improvement of chromatographic performance, J.ChromatognA, 1998, 797, 251-263. SAMPLE

Matrix: blood, tissue Sample preparation: Plasma, whole blood. Condition a Bond-Elut C8 SPE cartridge with MeOH and water. Mix plasma or whole blood and 50 mM pH 9 borate buffer, add to the SPE cartridge. Wash with water and MeCN. Elute with MeOH. Evaporate the eluate to dryness and reconstitute in 400 |xL mobile phase. Inject a 50fiL aliquot. Tissue. Homogenize (Braun micro-dismembrator) 100 mg tissue with 400 |xL water while frozen in liquid nitrogen, thaw, rinse twice with 250 JJLL 1 M pH 3 potassium phosphate buffer. Centrifuge at 2740 g at 20 for 20 min, separate supernatant (Sl). Extract pellet with 1 mL MeOH for 15 min with sonication. Centrifuge at 20 for 10 min, evaporate the supernatant to dryness under a stream of nitrogen at 40. Reconstitute residue in 500 jxL 15 mM pH 3 potassium phosphate buffer add to Sl, centrifuge. Suck sample slowly through a BondElut C8 SPE cartridge. Wash twice with water, elute twice with 200 |xL MeOH, evaporate the eluate to dryness under a stream of nitrogen at 40. Reconstitute in 500 |xL mobile phase. Inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 17 X 4 5 |m Spherisorb C6 Column: 150 X 4.6 5 fjim Spherisorb C6 Mobile phase: MeCN: 15 mM pH 3 potassium phosphate buffer 17:83 (plasma) or 10:90 (tissue, blood) Flow rate: 1 Injection volume: 50 Detector: UV 230
CHROMATOGRAM

Retention time: 4.4 (plasma), 6.8 (whole blood, tissue) Internal standard: atenolol
OTHER SUBSTANCES Extracted: sotalol KEYWORDS

whole blood; plasma; heart; SPE; atenolol is IS

REFERENCE Laer,S.; Neumann,J.; Scholz,H; Uebeler,R; Zimmermann,N. Determination of sotalol in human cardiac tissue by high-performance liquid chromatography, J.Chromatogr.B, 1996, 681, 291-298. SAMPLE

Matrix: blood, urine

Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JJLL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLCVARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 3.637 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: bulk Sample preparation: Dissolve 10 jxmole compound (as free base or hydrochloride) in 500 (xL MeCN, add 250 |xL 5% sodium carbonate (for hydrochlorides only), add 500 jxL 100 mM reagent in MeCN, vortex for 1 min, heat at 60 for 2 h, add 100 jjimole L-proline, heat at 60 for 30 min. Remove a 100 |xL aliquot and dilute it with mobile phase, neutralize with acetic acid, inject a 10 |xL aliquot. Prepare the reagent ((R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL (lR,2R)-(-)-l,2-diaminocyclohexane, 12 mL water, and 12 mL EtOH, heat the oil bath to 80, add 2.8 mL carbon disulfide dropwise (making sure that the product does not start to precipitate), when addition is complete reflux for 1 h, acidify with 500 jxL 5 M HCl, reflux for 12 h, cool, filter, wash the solid with a little cold EtOH to give trans-4,5-tetramethyleneimidazolidine-2-thione as a white fluffy solid (mp 148-150) (Tetrahedron 1993, 49, 4419). Stir 7.97 g 3,5-dinitrobenzoyl chloride in 30 mL dichloroethane at 50, add a solution of 6 g trans-4,5-tetramethyleneimidazolidine-2-thione in 120 mL dichloroethane containing a catalytic amount of 4-(dimethylamino)pyridine over 15 min, reflux for 2 h, remove the crystals of (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate by filtration, evaporate the filtrate to dryness and dissolve the residue in 60 mL dichloroethane, reflux for 16 h to obtain more (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexylisothiocyanate (mp >250, [a]546 = -133 (c = 1) in MeCN).
HPLC VARIABLES

Column: 125 X 4 5 |xm Lichrospher 60 RP Select B Mobile phase: MeCN:20 mM ammonium acetate 55:45

Flow rate: 1

Injection volume: 10 Detector: UV 254

CHROMATOGRAM

Retention time: k' 1.63, k' 2.24 (enantiomers)

OTHER SUBSTANCES

Also analyzed: acebutolol, alprenolol, carazolol, carvedilol, formoterol, methamphetamine, metipranolol, metoprolol, nifenanol, nitrilo atenolol, oxprenolol, pindolol, propranolol, xamoterol
KEYWORDS

derivatization; chiral
REFERENCE Kleidernigg,O.R; Posch,K.; Lindner,W. Synthesis and application of a new isothiocyanate as a chiral derivatizing agent for the indirect resolution of chiral amino alcohols and amines, J.ChromatognA, 1996, 729, 33-42. SAMPLE

Matrix: perfusate Sample preparation: Dilute perfusate 101 times with water and inject a 10 |xL aliquot.
HPLCVARIABLES

Column: ixBondapak C18 Mobile phase: MeCN:water:acetic acid 19:80:1 containing 625 nM 1-heptane-sulfonic acid Flow rate: 2 Injection volume: 10 Detector: F ex 225 em300
CHROMATOGRAM

Retention time: 2.1 Limit of quantitation: 400 ng/mL OTHER SUBSTANCES Extracted: metoprolol
KEYWORDS

rat
REFERENCE Lindahl,A.; Krondahl,E.; Gruden,A.-C; Ungell,A.-L.; Lennernas,H. Is the jejunal permeability in rats age-dependent?, Pharm.Res., 1997, 14, 1278-1281. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere C18 Mobile phase: MeOH:10 mM pH 3.5 sodium phosphate buffer 15:85 Flow rate: 1 Detector: UV 201
REFERENCE Walter,E.; Janich,S.; Roessler,B.J.; Hilfinger,J.M.; Amidon,G.L. HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: In vitro-in vivo correlation with permeability data from rats and humans, J.Pharm.ScL, 1996, 85, 1070-1076.

SAMPLE Matrix: solutions

HPLC VARIABLES

Guard column: Dynamax C18 (Rainin) Column: 250 X 4.6 5 jxm Dynamax 300AC18 (Rainin) Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 10:90:0.1. B was MeCN: water.trifluoracetic acid 80:20:0.1. A:B from 100:0 to 70:30 over 12 min Flow rate: 1 Detector: F ex 275 em 295
OTHER SUBSTANCES

Simultaneous: Tyr-containing peptides (F ex 278 em 305)

REFERENCE Sorensen,M.; Steenberg,B.; Knipp,G.T.; Wang,W.; Steffansen,B.; Frokjaer,S.; Borchardt,R.T. The effect of (3-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells, Pharm.Res., 1997,14, 1341-1348. SAMPLE

Matrix: solutions Sample preparation: Mix 300 |xL of a 30 JAM solution in dichloromethane with 10 |xL 20 mM l-(6-methoxy-2-naphthyl)ethyl isothiocyanate in anhydrous dichloromethane and 50 J L J L L 0.1% triethylamine in dichloromethane, vortex thoroughly, heat at 50 for 1.5 h, inject an aliquot. (Synthesize l-(6-methoxy-2-naphthyl)ethyl isothiocyanate as follows (protect from light). Dissolve 500 mg (S)-(+)-naproxen in 50 mL dry toluene, slowly add 5 mL freshly distilled thionyl chloride, reflux for 1 h, evaporate to dryness under vacuum, dry the acyl chloride (mp 87.5) under vacuum over KOH for 2 days. Dissolve 0.5 mmoles acyl chloride in 5 mL acetone, stir at 0, add 0.6 mmoles sodium azide dissolved in ice water, stir at 0 for 30 min, add 10 mL ice-cold water, filter, dry solid in a desiccator under vacuum. Dissolve the solid in 1 mL toluene or dichloromethane (dried over 3 A molecular sieve), reflux for 10 min, evaporate, store resulting isocyanate (mp 51) under vacuum over a desiccant. Dissolve 0.5 mmole isocyanate in 5 mL acetone, add 20 mL 8.5% phosphoric acid, heat to 80 for 1.5 h, adjust to pH 13, extract with diethyl ether:dichloromethane 4:1. Wash the organic layer twice with water, dry over anhydrous sodium sulfate, evaporate to dryness, dissolve in 1 mL toluene, evaporate to give the amine from naproxen as crystals (mp 53) (Pharm.Res. 1990, 7, 1262). Dissolve 1 mmole 1,1-thiocarbonyldiimidazole in 15 mL ice-cold chloroform, stir at 0, add dropwise 1 mmole of the amine dissolved in 10 mL chloroform, stir at room temperature for 1.5 h, evaporate to dryness, reconstitute with carbon tetrachloride (Caution! Carbon tetrachloride is a carcinogen!), filter, evaporate the filtrate to dryness, store the resulting oil in a desiccator, purify on a short silica gel column with dichloromethane:light petroleum 50:50 to give l-(6-methoxy2-naphthyl)ethyl isothiocyanate as a slightly yellow liquid (store in the freezer under argon).)
HPLCVARIABLES

Column: 250 X 4 5 |xm Zorbax ODS Mobile phase: MeCN.water 50:50 Flow rate: 1 Injection volume: 100 Detector: UV 230, F ex 270 em 350
CHROMATOGRAM

Retention time: k' 5.2 (S-(-)), 6.1 (R-(+)) OTHER SUBSTANCES Simultaneous: diacetolol

KEYWORDS

derivatization; chiral; F not much more sensitive than UV; a = 1.17

REFERENCE Btischges,R.; Linde,H.; Mutschler,E.; Spahn-Langguth,H. Chloroformates and isothiocyanates derived from 2-arylpropionic acids as chiral reagents: synthetic routes and chromatographic behaviour of the derivatives, J.ChromatogrA, 1996, 725, 323-334. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 10 (xm Partisil ODSl Mobile phase: MeOH:50 mM pH 3.0 phosphoric acid 10:90 Column temperature: 30 Flow rate: 1.5 Detector: radioactivity detection
OTHERSUBSTANCES

Also analyzed: cimetidine, hydrochlorothiazide, ranitidine

KEYWORDS

tritium labeled
REFERENCE Collett,A.; Sims,E.; Walker,D.; He,Y.-L.; Ayrton,J.; Rowland,M.; Warhurst,G. Comparison of HT29-18-d and Caco-2 cell lines as models for studying intestinal paracellular drug absorption, Pharm.Res., 1996, 13, 216-221. SAMPLE

Matrix: solutions
HPLC VARIABLES

Guard column: 10 X 3.2 5 fim Partisil ODS3 Column: 100 X 4.6 5 jim Partisil ODS3 Mobile phase: MeCN:buffer 10:90 (Buffer was 60 mM KH2PO4 adjusted to pH 3.0 with phosphoric acid.) Flow rate: 0.6-1 Injection volume: 10-100 Detector: UV 270 OTHER SUBSTANCES Also analyzed: practolol
REFERENCE Palm,K.; Luthman,K.; Ungell,A.-L.; Strandlund,G.; Artursson,P. Correlation of drug absorption with molecular surface properties, J.Pharm.ScL, 1996, 85, 32-39. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Vydac C18 Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic acid in MeCN. A:B from 95:5 to 65:35 over 9 min. Column temperature: 40

Flow rate: 1 Detector: UV (wavelength not given)

OTHERSUBSTANCES

Simultaneous: dexamethasone
REFERENCE Rubas,W.; Cromwell,M.E.M.; Shahrokh,Z.; Villagran,J.; Nguyen,T.-N.; Wellton,M.; Nguyen,T.-H.; Mrsny,R.J. Flux measurements across Caco-2 monolayers may predict transport in human large intestinal tissue, J.Pharm.ScL, 1996, 85, 165-169. SAMPLE

Matrix: solutions Sample preparation: Mix a 100 |xL of a 10 |xM solution in MeCNrwaterrtriethylamine 50: 50:0.1 with 100 JLJLL 1 mM (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole in MeCN, heat in the dark at 65 for 1.5 h, inject an aliquot. (Synthesis of (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole is as follows. Dissolve 0.5 g magnesium sulfate heptahydrate and 6 g NaOH in 60 mL water, throughout the reaction keep the flask at about 20 with cold water cooling, add 15 mL 30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl peroxide in one go, stir for 10 min, pour into 150 mL 20% sulfuric acid, extract three times with 50 mL portions of chloroform, determine peroxybenzoic acid concentration by iodometric titration (Tetrahedron 1967, 23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in chloroform to 7 g 2,6-difluoroaniline dissolved in 100 mL chloroform, stir at room temperature, when reaction is complete (iodometric titration) wash with 2% sodium thiosulfate, wash with 5% sodium carbonate, wash with water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure, recrystallize 2,6difluoronitrosobenzene from EtOH (mp 108.5-109.5). Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO at room temperature and add a solution of 3.91 g sodium azide in 85 mL DMSO dropwise, let stand for about 1 h, add to a large volume of water, extract with ether, dry the extracts over anhydrous sodium sulfate, evaporate to dryness under reduced pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole as a colorless oil (bp 837 12 mm Hg) (J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium chloride drying tube), stir at room temperature for 1 h, reflux for 2 h, cool, slowly pour into ice water, remove the organic layer, extract the aqueous layer with chloroform, combine the organic layer, wash, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, take up the residue in 5 mL benzene (Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of silica gel (100-200 mesh Kanto Chemical) with n-hexane:benzene 50:50, evaporate the appropriate fractions to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow needles (mp 64-66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at 0-10 and add 1 g dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise, adjust pH to 5 with 1 M HCl, concentrate to about 10 mL under reduced pressure, extract three times with 200 mL portions of diethyl ether, wash with water, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, chromatograph on a 500 X 20 column of silica gel with chloroform, isolate the appropriate fraction and re-chromatograph on the same column with ethyl acetate:benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as white needles (mp 124-125) (yield = 1% !). On a Merck no. 5714 60F254 TLC plate eluted with chloroform DBD-F has Rf 0.32 and lies between two other reaction products (Analyst 1989, 114, 413). It is also reported that DBD-F can be purchased from Tokyo Kasei. Cool a solution of 16.4 g (S)-(-)-l-benzyl-3-pyrrolidinol in 164 mL pyridine to +5, add 19.35 g p-toluenesulfonyl chloride, stir at +10 for 48 h, evaporate to dryness, chromatograph using dichloromethane:acetone 95:5 to obtain (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine (mp 68). Heat a solution of (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL anhydrous DMF to 65, add 33.5 g sodium azide (Caution! Sodium azide is highly toxic!), stir at 60 for 7 h, filter, evaporate the filtrate to dryness under reduced pressure, dissolve the residue in ethyl acetate, wash twice with water, dry

over anhydrous magnesium sulfate, evaporate to obtain (3R)-3-azido-l-(phenylmethyDpyrrolidine as an oil. Add 3.5 g 10% palladium on carbon under nitrogen to a solution of 7.05 g (3R)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8 m L I M HCl in water and 245 mL EtOH, hydrogenate at atmospheric pressure for 30 min, add 3.5 g catalyst, hydrogenate for 2 h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate to dryness under reduced pressure, take up the residue in 70 mL EtOH, filter, evaporate the filtrate to dryness under reduced pressure, repeat this operation twice, crystallize with the minimum amount of EtOH to obtain (3R)-3-aminopyrrolidine dihydrochloride (J. Med. Chem. 1992, 35, 4205). 3R-(+)-aminopyrrolidine is also reported to be available from Tokyo Kasei. Add 100 mg 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole in 20 mL MeCN dropwise to a stirred solution of 200 mg 3R-(+)-aminopyrrolidine in 20 mL MeCN at 010, stir at room temperature for 30 min, remove the MeCN by evaporation under reduced pressure, dissolve the residue in 50 mL 5% HCl, wash 3 times with 50 mL portions of ethyl acetate, adjust the pH of the aqueous solution to 13-14 with 5% NaOH, extract 6 times with 50 mL portions of ethyl acetate. Combine the organic layers and wash them with 20 mL water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure, recrystallize from hexane to obtain (R)-(-)-4-(3-aminopyrrolidin-l-yl)-7(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole as orange crystals (mp 96-98) (Analyst 1992, 117, 727). Add 100 fiL thiophosgene in 10 mL benzene (Caution! Benzene is a carcinogen!) to 100 mg (R)-(-)-4-(3-aminopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)2,1,3-benzoxadiazole in 100 mL acetone, reflux for 1 h, remove the solvent by evaporation under reduced pressure, suspend the residue in 100 mL water, extract 4 times with 25 mL portions of benzene. Combine the extracts and wash them with 20 mL water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure, recrystallize from hexane:benzene 1:2 to obtain (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole as yellow crystals (mp 160-170 d) (Analyst 1995, 120, 385).)
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Inertsil ODS-80A Mobile phase: MeCN:water:trifluoroacetic acid 35:65:0.1 Column temperature: 40 Flow rate: 1 Detector: F ex 460 em 550
CHROMATOGRAM

Retention time: 26.7, 32.8 (enantiomers) Limit of detection: 96-116 fmole

OTHER SUBSTANCES

Also analyzed: carteolol, timolol

KEYWORDS

derivatization; chiral
REFERENCE Toyo'oka,T.; Toriumi,M.; Ishii,Y. Enantioseparation of (5-blockers labelled with a chiral fluorescent reagent, R(-)-DBD-PyNCS, by reversed-phase liquid chromatography, J.Pharm.Biomed.AnaL, 1997,15, 1467-1476.

Atovaquone
Molecular formula: C22H19ClO3 Molecular weight: 366.84 CAS Registry No.: 95233-18-4 Merck Index: 898 SAMPLE

Matrix: blood Sample preparation: 100 |xL Plasma + 1 mL phosphate buffer + 5 mL hexanerisoamyl alcohol 98:2, shake for 15 min, centrifuge at 1000 g for 5 min. Remove 2-4 mL organic layer and add it to 1 mL isopropanol, evaporate under a stream of nitrogen, dissolve in 200 txL MeOH: 1% acetic acid 80:20, vortex for 10 s, inject a 50 JJLL aliquot.
HPLC VARIABLES

Guard column: 10 X 4 Keystone Cl guard column Column: 150 X 4.6 5 |xm Supelcosil LC-I Mobile phase: MeOH: 1% acetic acid 70:30 Flow rate: 1 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 7 Limit of quantitation: 250 ng/mL
OTHER SUBSTANCES

Simultaneous: pentamidine, zidovudine, trimethoprim, sulfamethoxazole

KEYWORDS

plasma
REFERENCE DeAngelis,D.V.; Long,J.D.; Kanics,L.L.; Woolley,J.L. High-performance liquid chromatographic assay for the measurement of atovaquone in plasma, J.Chromatogr.B, 1994, 652, 211-219. SAMPLE

Matrix: blood Sample preparation: 100 jxL Plasma + 1 mL 50 mM pH 7.0 KH2PO4ZNaOH buffer + 5 mL hexane:3-methyl-l-butanol 98:2, tumble mix for 11 min, centrifuge at 1500 rpm for 11 min. Remove a 2 mL aliquot of the organic layer and add it to 800 |xL isopropanol and 200 |xL 100 ng/mL IS in isopropanol, evaporate under reduced pressure at 5 psi at 50 for 11 min, reconstitute with 500 |xL hexane:3-methyl-l-butanol 98:2, evaporate for 11 min, reconstitute with 200 |xL MeOH: 1% pH 3.1 acetic acid 80:20, vortex for 15 s, inject a 50 jxL aliquot.
HPLC VARIABLES

Guard column: 10 X 2 reverse phase stainless steel column (Chrompack) Column: two 100 X 3 5 |xm Chromspher C8 glass columns in series (Chrompack) Mobile phase: MeCN:0.4% pH 2.0 trifluoroacetic acid 65:35 Flow rate: 0.6 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 9

Internal standard: trans-2-hydroxy-3-(4-phenylcyclohexyl)-l,4-naphthalenedione (59C80) (7) Limit of quantitation: 250 ng/mL

KEYWORDS

plasma
REFERENCE Studenberg,S.D.; Long,J.D.; Woolf,J.H.; Bruner,C.J.; Wilson,D.; Woolley,J.L. A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma, J.Pharm.Biomed.Anal., 1995, 13, 1383-1393. SAMPLE

Matrix: blood Sample preparation: Heat plasma at 56 for 1 h. 200 |xL Plasma + 50 JJLL 100 |xg/mL IS in MeOHiDMF 99:1, mix well, add 400 |xL MeCN:l% aqueous acetic acid 85:15, mix, centrifuge at 14000 g for 3 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Spherisorb C6 Mobile phase: MeOH:0.2% pH 2 aqueous trifluoroacetic acid containing 10 mM triethylamine 76:24 Column temperature: 25 Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 8.8 Internal standard: trans-2-hydroxy-3-(4-phenylcyclohexyl)-l,4-naphthalenedione (Burroughs-Wellcome) (7.4) Limit of detection: 500 ng/mL
OTHER SUBSTANCES

Simultaneous: clindamycin, pentamidine, pyrimethamine, trimethoprim Noninterfering: clarithromycin, dapsone, didanosine, fluconazole, folinic acid, foscarnet, gangiclovir, sulfadiazine, sulfamethoxazole, trimetrexate, zalcitabine, zidovudine
KEYWORDS

plasma
REFERENCE Hansson,A.G.; Mitchell,S.; Jatlow,R; Rainey,P.M. Rapid high-performance liquid chromatographic assay for atovaquone, J.Chromatogr.B, 1996, 675, 180-182.

Atracurium besylate
Molecular formula: C65H82N2O18S2 Molecular weight: 1243.50 CAS Registry No.: 64228-81-5 Merck Index: 900

SAMPLE

Matrix: blood Sample preparation: 250 |xL Plasma + 250 yiL picric acid (1:50 dilution of saturated picric acid solution) + 250 JJLL alcuronium solution + 250 |xL water + 2.5 mL dichloromethane: isopropanol 85:15, vortex for 15 s, centrifuge at 1500 g for 10 min. Remove the organic phase and evaporate it to dryness at 40 under a stream of nitrogen, reconstitute the residue in 150-250 \xL MeCN:water 40:60, centrifuge at 1500 g for 4 min, inject a 20-100 IxL aliquot.
HPLCVARIABLES

Column: 150 X 3.9 pPorasil Mobile phase: MeCN:2 mM sulfuric acid 50:50 Flow rate: 2 Injection volume: 20-100 Detector: UV 210
CHROMATOGRAM

Retention time: 3.5 Internal standard: alcuronium (4.5) Limit of detection: 25 ng/mL
OTHER SUBSTANCES

Also analyzed: tubocurarine, metocurine

KEYWORDS

plasma
REFERENCE Bjorksten,A.R.; Beemer,G.H.; Crankshaw,D.P. Simple high-performance liquid chromatographic method for the analysis of the non-depolarizing neuromuscular blocking drugs in clinical anaesthesia, J.Chromatogr., 1990, 533, 241-247. SAMPLE

Matrix: blood Sample preparation: Adjust pH of plasma to 4 with 2 M sulfuric acid. 250 JULL Acidified plasma + 10 (xL 0.5 M sulfuric acid + 50 |xL 5 (xg/mL verapamil in water, mix, add 600 |xL MeCN, vortex for 1 min, centrifuge at 2400 g for 10 min, inject a 50 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 100 X 4.9 5 |jum Spherisorb C8

Next Page

Mobile phase: Gradient. A was MeCN:MeOH:30 mM K2HPO4 37.5:5:57.5, adjust final pH to 5. B was MeCN:MeOH:100 mM K2HPO4 37.5:15:47.5, adjust final pH to 5. A:B 0:100 to 100:0 over 8 min. Flow rate: 1.7 Injection volume: 50 Detector: F ex 240 em 320 CHROMATOGRAM Retention time: 6.2 Internal standard: verapamil (4) Limit of detection: 20 ng/mL OTHER SUBSTANCES Simultaneous: laudanosine
KEY WORDS

plasma
REFERENCE

Varin,E; Ducharme,J.; Besner,J.G.; Theoret,Y. Determination of atracurium and laudanosine in human plasma by high-performance liquid chromatography, J.Chromatogr., 1990, 529, 319-327.
SAMPLE

Matrix: blood Sample preparation: Condition a Waters C18 SPE cartridge with 4 mL MeOH and 8 mL water. 500 |JLL Plasma + 2 mL 15 mM sulfuric acid, add a 500 (xL aliquot to the SPE cartridge, wash with 2 mL 5 mM PIC B-8, elute with 1.5 mL MeOH containing 5 mM PIC B-8, inject an aliquot of the eluate.
HPLC VARIABLES

Guard column: C-I (Keystone) Column: 150 X 4.6 Spherisorb C-I Mobile phase: Gradient. A was water containing 5 mM octanesulfonic acid (PIC B-8). B was MeCN:water containing 5 mM octanesulfonic acid (PIC B-8). A:B 52:48 for 8 min, then A:B 12:88 for 9.5 min (step gradient), re-equilibrate at initial conditions for 4.5 min (step gradient). Flow rate: 1 Detector: F ex 202 em 320
CHROMATOGRAM

Retention time: 15.5 (cis isomer) Limit of detection: <40 nM

OTHER SUBSTANCES

Extracted: metabolites, laudanosine

KEY WORDS

human; rat; plasma; SPE

REFERENCE

Welch,R.M.; Brown,A.; Ravitch,J.; Dahl,R. The in vitro degradation of cisatracurium, the R, cis-R'-isomer of atracurium, in human and rat plasma, Clin.Pharmacol.Ther., 1995, 58, 132-142.

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Atropine
Molecular formula: C17H23NO3 Molecular weight: 289.37 CAS Registry No.: 51-55-8, 52-88-0 (atropine methylnitrate) Merck Index: 907 LednicerNo.: 1 35, 71, 93; 2 71 SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 100 jxL 5 ng/mL scopolamine in MeOH, vortex briefly, add 50 |xL 1 M ammonium hydroxide, mix, add 5 mL dichloromethane, shake horizontally for 5 min, centrifuge at 2500 rpm for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |xL motile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Guard column: 10 X 2 5 jxm BDS C18 (Keystone) Column: 50 X 3 3 |xm BDS C18 (Keystone) Mobile phase: MeCN:MeOH:10 mM ammonium acetate 62.5:37.5:15 Flow rate: 0.5 Injection volume: 20 Detector: MS, Perkin Elmer Sciex API Ill-Plus triple quadrupole, APCI, nebulizer 400 and 80 psi, auxiliary nitrogen 1.2 L/min, curtain gas 1.2 L/min, interface 55, collision gas argon, electron multiplier 3000 V, declustering potential 35 V, collision energy 35 eV
CHROMATOGRAM

Retention time: 1.2 Internal standard: scopolamine (0.8) Limit of quantitation: 20 pg/mL
KEYWORDS

plasma; protect from light

REFERENCE Xu,A.; Havel,J.; Linderholm,K.; Hulse,J. Development and validation of an LC/MS/MS method for the determination of L-hyoscyamine in human plasma, J.Pharm.Biomed.AnaL, 1996, 14, 33-42. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 (xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) uL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min.

Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 10.388
KEYWORDS

whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: bulk, plants Sample preparation: Place 0.5 g powdered crude drug in 25 mL mobile phase, reflux 30 min, cool, centrifuge at 1600 g, decant wash residue twice with 10 mL portions of mobile phase, combine extracts and washings, make up to 50 mL with mobile phase, inject 10 IxL aliquot.
HPLCVARIABLES

Column: 150 X 4 5 |xm TSK gel 120A ODS Mobile phase: MeCN:67 mM pH 2.5 phosphate buffer 35:65 containing 17.5 mM sodium dodecylsulfate Column temperature: 35 Flow rate: 1.5 Injection volume: 10 Detector: UV 210 CHROMATOGRAM Retention time: 15 OTHER SUBSTANCES Simultaneous: scopolamine
REFERENCE Oshima,T.; Sagara,K.; Tbng,Y.Y.; Zhang,G.; Chen,Y.H. Application of ion-pair high performance liquid chromatography for analysis of hyoscyamine and scopolamine in solanaceous crude drugs, Chem.Pharm.BulKTokyo), 1989, 37, 2456-2458. SAMPLE

Matrix: formulations Sample preparation: Oral solutions. Dilute an amount oral solution equivalent to 25 jxg atropine sulfate to 10 mL with EtOH. Tablets. Weight out finely powdered tablets equivalent to 25 |xg atropine sulfate, add 10 mL MeCN:water 50:50, sonicate with frequent swirling for 15 min. Filter (0.45 |xm membrane filter), discard the first portion. Inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jjim Spherisorb CN Mobile phase: A:B 34:66 (Prepare A as follows. Dissolve 192 mg 1-pentanesulfonic acid sodium salt monohydrate in 200 mL water. Add 800 mL water and 1 mL orthophosphoric

acid. Prepare B as follows. Dissolve 192 mg 1-pentanesulfonic acid sodium salt monohydrate in 200 mL water. Add 800 mL MeCN and 1 mL orthophosphoric acid.) Flow rate: 1.7 Injection volume: 50 Detector: UV 220
CHROMATOGRAM Retention time: 4.8 OTHER SUBSTANCES

Simultaneous: metabolites, diphenoxylate

KEYWORDS

oral solutions; tablets

REFERENCE Lehr,G.J. Determination of diphenoxylate hydrochloride and atropine sulfate in combination drug formulations by liquid chromatography, JAOAC Int., 1996, 79, 1288-1293. SAMPLE

Matrix: formulations Sample preparation: Tablets, capsules. Powder tablets or remove contents of capsules, weigh out amount equivalent to about 600 |xg hyoscyamine sulfate-atropine sulfate, add 25 mL 25 mM sulfuric acid, shake for 15 min, centrifuge at 3000 rpm for 5 min. Remove 5 mL of the supernatant and extract it twice with 30 mL portions of dichloromethane, discard the organic phase, add 2 mL buffer to the aqueous phase, extract with four 30 mL portions of dichloromethane, filter extracts through dichloromethane-rinsed glass wool, add 3 mL 2.25 |xg/mL theophylline in dichloromethane, distil off the dichloromethane through a Snyder column by using a steam bath, when the volume reaches 10 mL rinse the column with 1-2 mL dichloromethane, continue distillation to 0.5-1 mL, remove the column and rinse the concentrator tube-column junction with 1 mL dichloromethane, evaporate to 1 mL with a stream of air at 40, add 100 |xL 1% concentrated HCl in MeOH, mix, evaporate to dryness with a stream of air at 40, rinse the sides of the concentrator tube with 500 jxL MeOH, evaporate to dryness with a stream of air at 40, dissolve the residue in 300 |xL water, inject a 20 \xL aliquot. Elixirs. Add an amount equivalent to about 600 |xg hyoscyamine sulfate-atropine sulfate to a 150 mL beaker, warm at 40 with a current of air for 30 min to remove alcohol, cool, make up to 25 mL with water, remove 5 mL of this solution, add 2 mL 100 mM sulfuric acid, extract twice with 30 mL portions of dichloromethane, discard the organic phase, add 2 mL buffer to the aqueous phase, extract with four 30 mL portions of dichloromethane, filter extracts through dichloromethane-rinsed glass wool, add 3 mL 2.25 |xg/mL theophylline in dichloromethane, distil off the dichloromethane through a Snyder column by using a steam bath, when the volume reaches 10 mL rinse the column with 1-2 mL dichloromethane, continue distillation to 0.5-1 mL, remove the column and rinse the concentrator tubecolumn junction with 1 mL dichloromethane, evaporate to 1 mL with a stream of air at 40, add 100 jxL 1% concentrated HCl in MeOH, mix, evaporate to dryness with a stream of air at 40, rinse the sides of the concentrator tube with 500 uX MeOH, evaporate to dryness with a stream of air at 40, dissolve the residue in 300 |xL water, inject a 20 |xL aliquot. (Buffer was 5.3 g anhydrous sodium carbonate and 4.2 g sodium bicarbonate in 100 mL water, pH 9.4. Pass dichloromethane through 75 g basic aluminum oxide, Brockmann Activity Grade 1, store over 25 g alumina/4 L.)
HPLC VARIABLES

Column: 250 X 4 5 \xm Spherisorb ODS Mobile phase: MeOH:buffer 250:525 (The 50 mM tetramethylammonium phosphate buffer was prepared from 500 mL water + 23 mL 20% tetramethylammonium hydroxide in MeOH + 10 mL concentrated phosphoric acid, adjust to pH 2.0 with concentrated phosphoric acid, make up to 1 L with water.)

Flow rate: 0.8 Injection volume: 20 Detector: UV 220

CHROMATOGRAM

Retention time: 7.5 Internal standard: theophylline (6.5)

OTHER SUBSTANCES

Simultaneous: phenobarbital, scopolamine, atropine

KEYWORDS

tablets; capsules; elixirs

REFERENCE Pennington,L.J.; Schmidt,W.F. Belladonna alkaloids and phenobarbital combination Pharmaceuticals analysis I: High-performance liquid chromatographic determinations of hyoscyamine-atropine and scopolamine, J.Pharm.ScL, 1982, 71, 951-953. SAMPLE

Matrix: formulations Sample preparation: Grind tablets to a fine powder, weigh out amount containing 25 (xg atropine, extract with 40 mL chloroform, filter, wash filter with chloroform, make up filtrate to 50 mL with chloroform, mix. Remove a 1 mL aliquot and evaporate it to dryness under reduced pressure at 40, reconstitute with 500 |xL 4 mg/mL quinuclidine in acetone, add 500 |xL 2 mg/mL 1-anthroylnitrile in acetone, heat at 30 for 10 min, add 1 mL 2% phosphoric acid, cool to room temperature, make up to 10 mL with acetone, inject a 10 |xL aliquot. (1-Anthroylnitrile is available from Wako Chemicals, Richmond VA. Synthesis is as follows. Dissolve 50 g benzanthrone in 500 mL concentrated sulfuric acid with gentle warming, pour this solution cautiously into 4 L hot water with vigorous stirring. Boil the suspension and slowly add 200 g chromium(VI) oxide (Caution! Chromium oxide is a carcinogen and highly corrosive!), after 6 h cool the mixture, filter, wash the precipitate with hot water. Dissolve the precipitate in dilute ammonia and precipitate with acid, crystallize from boiling concentrated nitric acid to give anthraquinone-1-carboxylic acid (Ber. 1924, 57, 1775). Warm, on a water bath, anthraquinone-1-carboxylic acid in dilute ammonia with twice the amount of zinc dust, when the reaction has ceased (30 min ?) filter the reaction the reaction mixture, add HCl to the filtrate to obtain anthracene-1carboxylic acid as yellow needles, recrystallize from EtOH (mp 245) (Ber 1897, 30,1118). Stir 1 g anthracene- 1-carboxylic acid in 15 mL anhydrous dichloromethane, add 2 mL oxalyl chloride, reflux for 1 h, evaporate to give 1-anthroyl chloride as an oily residue. Dissolve 1-anthroyl chloride in 15 mL dichloromethane, add 3 mL trimethylsilyl cyanide, add 1 mg zinc iodide, stir at room temperature for 2 h, evaporate to dryness, recrystallize from hexane/dichloromethane to give 1-anthroylnitrile as orange-yellow needles (mp 1645) (Anal.Chim.Acta 1983, 147, 397).)
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Cosmocil 5C-18 (Nacalai Tesque, Tokyo) Mobile phase: MeCN:buffer 60:40 (Buffer was 20 mM sodium dodecyl sulfate adjusted to pH 3.5 with phosphoric acid.) Column temperature: 40 Flow rate: 1 Injection volume: 10 Detector: F ex 255 em 474
CHROMATOGRAM

Retention time: 12 Limit of detection: 10 ng/mL Limit of quantitation: 50 ng/mL

OTHER SUBSTANCES

Noninterfering: albumin, amomum seed, caffeine, chlorpheniramine, cinnamon bark, cloves, fennel, geranium herb, glycyrrhiza, lysozyme, swertia herb, thiamine, riboflavin
KEYWORDS

derivatization; tablets
REFERENCE Takahashi,M.; Nagashima,M.; Shigeoka,S.; Nishijima,M.; Kamata,K. Determination of atropine in pharmaceutical preparations by liquid chromatography with fluorescence detection, J.Chromatogr.A, 1997, 775, 137-141.

SAMPLE Matrix: plants Sample preparation: Dissolve alkaloids in 1 mL MeOH, add 40 ng homatropine, inject aliquot.
HPLCVARIABLES

Column: 150 X 4.1 5 |xm Hamilton PRP-I Mobile phase: MeCN: 100 mM pH 10.4 ammonium acetate Flow rate: 1 Injection volume: 20 Detector: MS thermospray, VG Trio-2, ion source 150, vaporizer tip 170, repeller electrode 150 V, m/z 290
CHROMATOGRAM

Internal standard: homatropine (m/z 276) Limit of detection: 2.5 ng/mL OTHER SUBSTANCES Simultaneous: scopolamine
KEYWORDS

total run time 6 min

REFERENCE Auriola,S.; Martinsen,A.; Oksman-Caldentey,K.M.; Naaranlahti,T. Analysis of tropane alkaloids with thermospray high-performance liquid chromatography-mass spectrometry, J.Chromatogr., 1991,562, 737-744.

SAMPLE Matrix: plants Sample preparation: Extract 0.1 g dry plant material with 10 mL MeOH for 10 min under reflux, filter, inject aliquot.
HPLC VARIABLES

Guard column: 40 X 4 10 |xm Hypersil ODS Column: 250 X 4 10 |im Hypersil ODS Mobile phase: MeOH:water 45:55 containing 0.1% phosphoric acid adjusted to pH 7 with triethylamine Flow rate: 1 Detector: UV 229 CHROMATOGRAM Retention time: 20.7

OTHER SUBSTANCES Simultaneous: scopolamine

REFERENCE Hagemann,K.; Piek,K.; Stockigt,J.; Weiler,E.W. Monoclonal antibody-based enzyme immunoassay for the quantitative determination of the tropane alkaloid, scopolamine, Planta Med., 1992, 58, 68-72.

SAMPLE Matrix: plants Sample preparation: 100 mg Freeze-dried powdered plant leaves + 10 mL mobile phase, heat at 40 for 15 min, filter, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4 4 jjim Novapack C18 Mobile phase: MeCNrwater 12.5:87.5 with 0.3% phosphoric acid adjusted to pH 2.2 with triethylamine Flow rate: 0.8 Injection volume: 20 Detector: UV 204
CHROMATOGRAM

Retention time: 11.7 Limit of detection: 50000 ng/g

OTHER SUBSTANCES

Simultaneous: tropic acid, scopolamine

REFERENCE Fliniaux,M.-A.; Manceau,E; Jacquin-Dubreuil,A. Simultaneous analysis of 1-hyoscyamine, 1-scopolamine and dl-tropic acid in plant material by reversed phase high-performance liquid chromatography, J.Chromatogr., 1993, 644, 193-197. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject a 10 [xh aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 |xm fiBondapak C18 Mobile phase: MeOHtacetic acid:triethylamine:water 20:1.5:0.5:78 Flow rate: 1.5 Injection volume: 10 Detector: UV CHROMATOGRAM Retention time: k' 4.76
REFERENCE Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418.

Auranofin
Molecular formula: C20H34AuO9PS Molecular weight: 678.49 CAS Registry No.: 34031-32-8 Merck Index: 911

SAMPLE

Matrix: solutions Sample preparation: Filter (0.45 or 0.22 |xm), inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.5 5 |xm octadecylsilane (Jones Chromatography) Mobile phase: MeOH:0.25% ammonium dihydrogen phosphate 65:35 Flow rate: 0.8 Injection volume: 20-50 Detector: UV 214
CHROMATOGRAM

Retention time: 7.5 Limit of detection: 0.2 ppm

KEY WORDS

Krebs-Ringer bicarbonate buffer

REFERENCE Tepperman,K; Finer,R.; Donovan,S.; Elder,R.C; Doi,J.; Ratliff,D.; Ng,K. Intestinal uptake and metabolism of auranofin, a new oral gold-based antiarthritis drug, Science, 1984, 225, 430-432. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Spherisorb ODS-2 Mobile phase: MeOH.water 50:50 containing 10 mM tetrabutylammonium chloride and 25 mM ammonium formate, pH 6.3 Column temperature: 30 Flow rate: 1 Detector: UV 227 or MS, Sciex Elan 250 ICP-MS, monitor gold 197, RF power 1.4 kW, nebulizer Ar gas flow rate 1 L/min, nebulizer spray chamber 17 CHROMATOGRAM Retention time: 18 Limit of detection: 0.3 ng
OTHER SUBSTANCES

Also analyzed: myochrysine

REFERENCE Zhao,Z.; Jones,W.B.; Tepperman,K.; Dorsey,J.G.; Elder,R.C. Determination of gold-based antiarthritis drugs and their metabolites in urine by reversed-phase ion-pair chromatography with ICP-MS detection, J.Pharm.Biomed.AnaL, 1992, 10, 279-287.

SAMPLE

Matrix: urine Sample preparation: Filter (0.45 |xm), inject a 200 jxL aliquot.
HPLC VARIABLES

Guard column: 50 X 4.6 5 jim Spherisorb ODS2 Column: 150 X 4.6 5 |xm B & J OD5 octadecyl Mobile phase: MeOHrwater 50:50 containing 10 mM tetrabutylammonium chloride and 25 mM ammonium formate, pH 6 Column temperature: 30 Flow rate: 1 Injection volume: 200 Detector: MS, Sciex Elan 250 ICP-MS, monitor gold 197, RF power 1.4 kW, nebulizer Ar gas flow rate 1 L/min, nebulizer spray chamber 17
CHROMATOGRAM

Retention time: 18 Limit of detection: 500 ng/mL

REFERENCE Zhao,Z.; Jones,W.B.; Tepperman,K.; Dorsey,J.G.; Elder,R.C. Determination of gold-based antiarthritis drugs and their metabolites in urine by reversed-phase ion-pair chromatography with ICP-MS detection, J.Pharm.BiomedAnaL, 1992, 10, 279-287.

SAMPLE Matrix: urine

Sample preparation: Rabbit urine. Centrifuge at 1000 g at 3 for 1 min, inject an aliquot. Human urine. Inject directly.
HPLC VARIABLES

Column: 150 X 4.6 YMC AM-302 octadecylsilyl (YMC) Mobile phase: MeOH:water 65:35 Flow rate: 1 Injection volume: 100 Detector: UV 412 with post-column reaction detection. Reagent was 50 J U L M 5,5'-dithiobis(2nitrobenzoic acid), 300 mM KI, and 50 mM pH 7.4 phosphate buffer delivered at 0.5 mL/ min, mixed with column effluent, passed though a 0.5 mm X 5 m PTFE reaction coil at 60.
CHROMATOGRAM Retention time: 6 KEYWORDS

human; rabbit; post-column reaction

REFERENCE Kizu,R.; Kaneda,M.; Yamauchi,Y; Miyazaki,M. Determination of auranofin, a chrysotherapy agent, in urine by HPLC with a postcolumn reaction and visible detection, Chem.Pharm.BulL, 1993, 41, 12611265.

Avermectin
Molecular formula: C48H74O14 Molecular weight: 875.11 Merck Index: 919

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, na-

lorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994, 18, 233-242.

Avitriptan
Molecular formula: C22H30N6O3S Molecular weight: 458.59 CAS Registry No.: 151140-96-4, 171171 -42-9 (fumarate)

SAMPLE

Matrix: blood Sample preparation: Add 50|xL 1 M pH 5 ammonium acetate and IS to 500 |xL plasma, mix. Add to a conditioned carboxylic acid BondElut SPE cartridge. Wash with pH 5.0 ammonium acetate and dichloromethane. Elute with 2 mL 1% triethylamine in MeOH. Evaporate, reconstitute the residue with 200|xL mobile phase, inject a 100 jxL aliquot
HPLC VARIABLES

Column: 250 X 4.6 DeltaBond CN (Keystone Scientific, Bellafonte, PA) Mobile phase: MeCN:MeOH:water 5:5:90 containing 10 mM pH 3 ammonium phosphate dibasic and 10 mM pH 3 tetramethylammonium hydroxide Flow rate: 1 Injection volume: 100 Detector: UV 287
CHROMATOGRAM

Retention time: 6.5 Internal standard: BMY-46317 (9) Limit of quantitation: 10 ng/mL
KEYWORDS

pharmacokinetics; radiolabeled; SPE; rat; plasma

REFERENCE Marathe,P.H.; Greene,D.S.; Barbhaiya,R.H. Disposition of [14C]avitriptan in rats and humans, Drug Metab.Dispos., 1997, 25, 881-888.

Azacyclonol
Molecular formula: C18H21NO Molecular weight: 267.37 CAS Registry No.: 115-46-8, 1798-50-1 (HCI) Merck Index: 925 LednicerNo.: 1 47 SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 1.9 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,

protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Azaperone
Molecular formula: C19H22FN3O Molecular weight: 327.40 CAS Registry No.: 1649-18-9 Merck Index: 931 Lednicer No.: 2 300

SAMPLE

Matrix: tissue Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and 5 mL water. Homogenize kidney with a kitchen grinder. Weigh out a 5 g sample and add 20 mL MeCN with continuous gentle mixing, mix vigorously on a vibromixer at 1500 rpm for 30 s, sonicate for 2 min, centrifuge at 4000 g for 5 min. Mix 7.5 mL sample extract and 40 mL 10% NaCl and add to SPE cartridge, wash with 1 mL 10 mM sulfuric acid, wash with 2 mL air, elute with 2 mL acidic MeCN. Place eluate in a washed tube and evaporate to 300 |L at 70 under a stream of nitrogen, mix gently, add 1 mL n-hexane, mix on a vibromixer for 30 s, centrifuge at 2000 g, inject a 50 |xL aliquot of the aqueous phase. (Acidic MeCN was 1 mL 50 mM sulfuric acid and 100 mL MeCN. The washed tube was prepared by rinsing with concentrated ammonia, water, and acetone and drying under a stream of nitrogen.)
HPLC VARIABLES

Guard column: 10 X 2.1 37-50 |xm Bondapak C18 Column: 300 x 3.9 Bondapak C18 Mobile phase: MeCN:water 55:45 containing 2.46 g/L anhydrous sodium acetate, pH adjusted to 6.5 with acetic acid Flow rate: 1.2 Injection volume: 50 Detector: UV 240 CHROMATOGRAM Retention time: 7.5 Limit of detection: 1 ng/g
OTHER SUBSTANCES

Extracted: azaperol, carazolol, acepromazine, xylazine, haloperidol, propiomazine, chlorpromazine

KEY WORDS

SPE; pig; kidney

REFERENCE Keukens,H-L; Aerts,M.M.L. Determination of residues of carazolol and a number of tranquilizers in swine kidney by high-performance liquid chromatography with ultraviolet and fluorescence detection, J.Chromatogr., 1989, 464, 149-161. SAMPLE

Matrix: tissue Sample preparation: Condition a Bond-Elut C18 SPE cartridge with 5 mL MeOH and 5 mL water. Cut pig kidney or liver into small pieces and homogenize. 5 g Homogenate + 10 mL MeCN, shake, vortex for 30 s, sonicate for 3 min, vortex for 30 s, sonicate for 3 min, centrifuge at 10000 g for 20 min. Add 7.5 mL supernatant + 40 mL 10% NaCl to the SPE cartridge at about 1 mL/min, do not allow cartridge to dry out, wash with 850 IxL 10 mM sulfuric acid, dry with air, elute with 3.5 mL acidic MeCN. Evaporate the

eluate to dryness under a stream of nitrogen at 50, reconstitute the residue in 300 |xL 10 mM sulfuric acid, vortex briefly, add 1 mL hexane, vortex for 30 s, centrifuge at 2000 g for 5 min, inject an aliquot of the aqueous layer. (Acidic MeCN was 1 mL 50 mM sulfuric acid in 100 mL MeCN.)
HPLC VARIABLES

Guard column: Hypersil 5 |xm SAS Cl Column: 250 mm long 5 jxm Hypersil SAS Cl Mobile phase: MeCN:water 50:50 containing 0.77 g/L ammonium acetate Flow rate: 2 Detector: E, ESA Model 5100A Coulochem, first electrode +0.4 V, second electrode (which was monitored) +0.7 V, Model 5020 guard cell after pump but before injector at +0.75 V CHROMATOGRAM Retention time: 6.5 Limit of detection: 2 ng/g
OTHER SUBSTANCES

Extracted: azaperol, chlorpromazine

KEYWORDS

acepromazine,

carazolol, xylazine, haloperidol,

propriomazine,

SPE; pig; kidney; liver

REFERENCE Rose,M.D.; Shearer,G. Determination of tranquilisers and carazolol residues in animal tissue using highperformance liquid chromatography with electrochemical detection, J.Chromatogr., 1992, 624, 471477. SAMPLE

Matrix: urine Sample preparation: Adjust 75 mL urine to pH 10 with 10 mL saturated borate buffer and concentrated ammonium hydroxide, extract with 100 mL dichloromethanerisopropanol 96:4. Extract the organic layer with 100 mL 100 mM HCl, adjust the pH of the aqueous layer to 10, extract the aqueous layer with 100 mL hexane:isopropanol 96:4. Dry the extract at 50 under a stream of nitrogen, reconstitute the residue in 500 JJIL MeOH, inject an aliquot.
HPLCVARIABLES

Column: 250 X 2.1 5 fim Suplex pKb-100 (Supelco) Mobile phase: Gradient. MeCN:50 mM pH 10 ammonium acetate from 20:80 to 100:0 over 20 min. Flow rate: 0.2 Detector: MS, Sciex API III triple quadrupole LC-MS-MS, heated nebulizer interface at 400, corona discharge current 3 |xA, orifice diameter 125 |xm, collision induced dissociation using argon, argon curtain thickness was 500 X 1012 molecules/cm2, collision energy 50 eV, positive ion mode
CHROMATOGRAM Retention time: 20.1 OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

horse; LC-MS

REFERENCE Chui,Y.C; Esaw,B.; Laviolette,B. Investigation of the metabolism of azaperone in the horse, J.Chromatogr.B, 1994, 652, 23-33.

Azasetron
Molecular formula: C17H20CIN3O3 Molecular weight: 349.82 CAS Registry No.: 123040-69-7, 123040-16-4 (HCI) Merck Index: 933

SAMPLE

Matrix: blood Sample preparation: Add 20 |JLL 2 M NaOH and 2 mL dichloroethane to 200 |xL serum. Shake for 10 min, centrifuge at 3000 rpm for 5 min. Remove a 1.6 mL portion of the upper organic layer, add 80 fxL 100 mM HCl, and 1.5 mL hexane, centrifuge at 3000 rpm for 5 min. Remove the upper organic layer, inject a 50 |xL aliquot of the lower layer.
HPLC VARIABLES

Column: 150 X 4 SenshuPak ODS-0151-N Mobile phase: MeCN:THF:100 mM pH 5 ammonium acetate 9.8:6.2:84 Column temperature: 50 Flow rate: 1 Injection volume: 50 Detector: F ex 318 em 382
KEYWORDS

rabbit; serum; pharmacokinetics

REFERENCE Moriyama,Y.; Arimori,K.; Nakano,M. Absorption characteristics of azasetron from rectal and oral routes in rabbits, BioLPharm.BulL, 1997, 20, 701-703.

Azatadine
Molecular formula: C20H22N2 Molecular weight: 290.41 CAS Registry No.: 3964-81-6, 3978-86-7 (maleate) Merck Index: 934 Lednicer No.: 2 424

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 jxm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acidrbuffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 10.70 (A), 4.55 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone

KEYWORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 200 (xL 5 jxg/mL 8-chloroazatadine in water + 1 mL 1 M NaOH + 6 mL diethyl ether, shake in a reciprocal shaker for 10 min, centrifuge at 1600 g for 5 min, freeze in dry ice/acetone. Remove the organic layer and add it to 500 JJLL 50 mM sulfuric acid, shake on a reciprocal shaker for 10 min, centrifuge at 1600 g for 5 min, freeze in dry ice/acetone, discard the organic layer. Add the aqueous layer to 1 mL 1 M NaOH, extract with 6 mL hexane, centrifuge, freeze in dry ice/acetone. Remove the organic layer and add it to 100 |xL concentrated ammonium hydroxide. Evaporate to dryness under a stream of nitrogen at 45, dissolve the residue in 500 |xL mobile phase, inject a 200 jxL aliquot.
HPLC VARIABLES

Guard column: silica Guard-Pak (Waters) Column: 300 X 3.9 10 |xm ixBondapak CN Mobile phase: MeCN:50 mM pH 7.5 KH2PO4 90:200 Flow rate: 2 Injection volume: 200 Detector: UV 214
CHROMATOGRAM

Retention time: 7.5 Internal standard: 8-chloroazatadine (10.5) Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES

Extracted: metabolites Simultaneous: chlorpheniramine, brompheniramine Noninterfering: pseudoephedrine, phenylpropanolamine

REFERENCE Alton,K.B.; Petruzzi,R.R; Patrick,J.E. High-performance liquid chromatographic assay for azatadine in human urine, J.Chromatogr., 1987, 385, 249-259.

Azathioprine
Molecular formula: C9H7N7O2S Molecular weight: 277.27 CAS Registry No.: 446-86-6 Merck Index: 935 Led nicer No.: 2 464

SAMPLE

Matrix: blood Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 2.5 mL MeOH and 5 mL 0.2% acetic acid. Mix 1 mL plasma with 40 jxL saturated EDTA solution, add to the SPE cartridge. Wash with 2 mL 0.2% acetic acid, centrifuge at 2200 g for 5 min to remove excess water, elute with 2 mL MeOH, evaporate to dryness under a stream of nitrogen at 37. Reconstitute the residue with 200 jxL mobile phase, vortex for 30 s, centrifuge at 11000 g for 5 min, inject an 80 (xL aliquot of the supernatant. (Prepare saturated EDTA solution by vortexing 2.5 g disodium (?) EDTA in 25 mL water for 5 min.)
HPLC VARIABLES

Guard column: 12.5 X 4 Zorbax ODS Column: 200 X 4.6 5 fjim Hypersil ODS Mobile phase: Gradient. A was MeCN: 1 mM triethylamine 0.8:99.2, adjusted to pH 3.2 with phosphoric acid. B was MeCN: 1 mM triethylamine 20:80, adjusted to pH 3.2 with phosphoric acid. A:B 100:0 for 5 min, to 50:50 over 2.5 min, maintain at 50:50 for 11.5 min to 100:0 over 2 min, re-equilibrate at initial conditions for 9 min Flow rate: 1.5 Injection volume: 80 Detector: UV 340
CHROMATOGRAM

Retention time: 15 Limit of quantitation: 5 ng/mL OTHER SUBSTANCES Extracted: metabolites

KEY WORDS

SPE; plasma; pharmacokinetics

REFERENCE Van Os,E.C; McKinney,J.A.; Zins,B.J.; Mays,D.C.; Schriver,Z.H.; Sandborn,W.J.; Lipsky,J.J. Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography, J.Chromatogr.B, 1996, 679, 147-154. SAMPLE

Matrix: blood Sample preparation: 500 |xL Serum + 25 ng 9-methylazathioprine + 4.5 mL ethyl acetate, vortex for 1 min, centrifuge for 1 min, repeat extraction. Combine the organic layers and evaporate them to dryness under reduced pressure at 35, reconstitute the residue in 250 jxL mobile phase, vortex for 10 s, sonicate for 10 min, inject a 200 JJLL aliquot.
HPLC VARIABLES

Guard column: 4 X 4.6 5 |xm LiChrospher 100 RP 18 Column: 250 X 4.6 5 |xm LiChrospher 60 Rp-select B

Mobile phase: MeCN:10 mM pH 2.3 potassium phosphate buffer 12:88 (Flush with MeCN: buffer 50:50 for 2 min after each run.) Column temperature: 22 Flow rate: 1 Injection volume: 200 Detector: UV 285
CHROMATOGRAM

Retention time: 16 Internal standard: 9-methylazathioprine (Add 400 mg anhydrous potassium carbonate and 200 (xL methyl iodide to a solution of 220 mg azathioprine in 7 mL DMF at 0-5, stir under nitrogen at 24 h, add 14 mL water, neutralize with 1 M HCl and sodium bicarbonate solution, filter, wash the solid with water, dry under vacuum to give 9-methylazathioprine (mp 174-5). Purify by precipitating from DMF solution with water.) (29) Limit of quantitation: 2.5 ng/mL OTHER SUBSTANCES Noninterfering: 6-mercaptopurine
KEYWORDS

serum; pharmacokinetics
REFERENCE Binscheck,T.; Meyer,H; Wellhoner,HH- High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples, J.Chromatogr.B, 1996, 675, 287-294.

Azelaic acid
Molecular formula: C9H16O4 Molecular weight: 188.22 CAS Registry No.: 123-99-9 Merck Index: 938

SAMPLE

Matrix: blood Sample preparation: Condition a 500 mg Bond Elut NH2 SPE cartridge with 3 mL MeOH and 12 mL 100 mM pH 7 NaH2PO4 buffer. Add 1 mL plasma to the SPE cartridge, wash with 3 mL water, wash with 3 mL MeCN, dry under vacuum, elute with 1 mL 500 mM formic acid in MeCN. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with 100 |xL 50 mM triethylamine in MeCN, vortex for 1 min, add 50 |xL 60 mM ethyl chloroformate in MeCN, vortex for 1 min, add 200 |JLL 3 mM L-leucine-(4-methyl-7coumarinylamide) in MeOH, vortex for 1 min, let stand for 4 min, evaporate to dryness under a stream of nitrogen, reconstitute with 200 jxL MeCN:water 50:50, inject a 5-15
JJLL aliquot. HPLC VARIABLES

Column: 250 X 4.6 5 ixm Axxiom octyl (Richard Scientific, Novato) Mobile phase: Gradient. A was 1 L water containing 2 mL 85% phosphoric acid. B was 1 L MeCN containing 2 mL 85% phosphoric acid. A:B 55:45 for 15 min, to 10:90 over 15 min. Injection volume: 5-15 Detector: F ex 330 em 390
CHROMATOGRAM Retention time: 30 OTHER SUBSTANCES

Extracted: dodecanedioic acid, hexadecanedioic acid, pimelic acid, tetradecanedioic acid

KEY WORDS

plasma; derivatization; SPE; dog; human

REFERENCE Levai,E; Liu,C.-M.; Tse,M.M.; Lin,E.T. Pre-column fluorescence derivatization using leucine-coumarnylamide for HPLC determination of mono- and dicarboxylic acids in plasma, Ada Physiol.Hung., 1995, 83, 39-46. SAMPLE

Matrix: blood, feces, urine Sample preparation: Serum. 1 mL Serum + 50 |xg sebacic acid, acidify to pH 1 with 1 M HCl, saturate with NaCl, extract three times with 10 mL warm (40) ethyl acetate. Combine the extracts and dry them over anhydrous sodium sulfate, evaporate to dryness under vacuum below 40, take up the residue in 1 mL MeCNiMeOH 80:20, add 3 mg of p-bromophenacyl bromide in MeCN, add 6 |xL N,N-diisopropylethylamine, heat at 50-60 for 10-15 min, evaporate some solvent, add reaction mixture to a TLC plate (Carlo Erba Stratocrom SI-AP, 0.25 mm silica gel, activated at 120 for 20 min), develop plate in benzene:hexane 3:1 (CAUTION! Benzene is a carcinogen!), remove material remaining at origin, extract three times with 1 mL MeCN, evaporate solvent to 500 |xL, inject a 20-50 |JLL aliquot. Urine, feces. 20-50 |xL Urine or feces diluted with water + 50 |xg sebacic acid, acidify to pH 1 with 1 M HCl, extract five times with 3 volumes of warm (40) ethyl acetate. Combine the extracts and dry them over anhydrous sodium sulfate, evaporate to

dryness under vacuum below 40, take up the residue in 1 mL MeCNrMeOH 80:20, add 3 mg of p-bromophenacyl bromide in MeCN, add 6 |xL N,N-diisopropylethylamine, heat at 50-60 for 10-15 min, evaporate some solvent, add reaction mixture to a TLC plate (Carlo Erba Stratocrom SI-AP, 0.25 mm silica gel, activated at 120 for 20 min), develop plate in benzene:hexane 3:1 (CAUTION! Benzene is a carcinogen!), remove material remaining at origin, extract three times with 1 mL MeCN, evaporate solvent to 500 jxL, inject a 20-50 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 5 jxm RP 18 (Brownlee) Mobile phase: Gradient. MeCNrwater adjusted to pH 3.10 with phosphoric acid 60:40 for 5 min then to 100:0 over 60 min. Column temperature: 40 Flow rate: 1 Injection volume: 20-50 Detector: UV 255
CHROMATOGRAM

Retention time: 32.2 Internal standard: sebacic acid (35.9) Limit of detection: 0.5 ng
OTHER SUBSTANCES

Extracted: other dicarboxylic acids (C4-C13)

KEYWORDS

serum; human; rat

REFERENCE Passi,S.; Nazzaro-Porro,M.; Picardo,M.; Mingrone,G.; Fasella,P. Metabolism of straight saturated medium chain length (C9 to C12) dicarboxylic acids, J.LipidRes., 1983, 24, 1140-1147. SAMPLE

Matrix: follicles, skin Sample preparation: Wash skin with 500 JJLL acetone, homogenize follicles in acetone, centrifuge at 13400 g for 5 min, remove a 400 (xL portion of the supernatant, re-centrifuge. Evaporate a 30 |xL aliquot, take up the residue in 1 mL MeCN:MeOH 80:20, add 3 mg of p-bromophenacyl bromide in MeCN, add 6 JJLL N,N-diisopropylethylamine, heat at 5060 for 10-15 min, evaporate some solvent, add reaction mixture to a TLC plate (Carlo Erba Stratocrom SI-AP, 0.25 mm silica gel, activated at 120 for 20 min), develop plate in benzeneihexane 3:1 (CAUTION! Benzene is a carcinogen!), remove material remaining at origin, extract three times with 1 mL MeCN, evaporate solvent to 50 jxL, inject a 2050 |JLL aliquot.
HPLCVARIABLES

Guard column: 24 X 3.9 Bondapak C18 Column: 150 X 3.9 Novapak C18 Mobile phase: Gradient. MeCN:water adjusted to pH 3.10 with phosphoric acid 60:40 for 5 min then to 100:0 over 60 min. Column temperature: 40 Flow rate: 1 Injection volume: 20-50 Detector: UV 254
CHROMATOGRAM

Limit of detection: 50 ng/mL

REFERENCE Bojar,R.A.; Cutcliffe,A.G.; Graupe,K.; CunlifFe,W.J.; Holland,K.T. Follicular concentrations of azelaic acid after a single topical application, Br.J.DermatoL, 1993, 129, 399-402. SAMPLE

Matrix: formulations Sample preparation: Condition a 1 g 100 jxm Bakerbond Florisil SPE cartridge with 5 mL THFrhexane 40:60. Condition a 500 mg 40 \ym Bakerbond C18 SPE cartridge with MeCN and MeCN:water 65:35. 200 mg Cream + 3 mL THF:hexane 40:60, sonicate, centrifuge at 3000 rpm for 2 min, repeat extraction twice. Add the supernatants to the Florisil SPE cartridge, wash with 2 mL THF:hexane 40:60, dry under vacuum, elute with two 2 mL portions of hexane:isopropanol 70:30. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 10 mL MeOH, neutralize (phenolphthalein endpoint) with 0.01% KOH in MeOH, evaporate to dryness under a stream of nitrogen at room temperature, reconstitute with 15 mL MeCN, add 5 mL 2 mM p-bromophenacyl bromide in MeCN containing 100 JJLM 18-crown-6, add 10 mL MeCN, stir at 80 for 30 min, cool to 4, dilute 1:50 with MeCN:water 65:35, add a 1 mL aliquot to the C18 SPE cartridge, wash with two 3 mL aliquots of MeCN:water 75:25, elute with 10 mL MeCN. Remove a 1 mL aliquot of the eluate and add it to 100 jxL 85.125 jxg/mL sebacic acid, inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4 5 |xm LiChrospher 100-RP-18 Mobile phase: MeCN:water 75:25 Flow rate: 1 Injection volume: 5 Detector: UV 254
CHROMATOGRAM

Retention time: 6.06 Internal standard: sebacic acid (7.59)

KEY WORDS

derivatization; cream; SPE

REFERENCE FeriolijV.; Rustichelli,C; Vezzalini,R; Gamberini,G. Determination of azelaic acid in Pharmaceuticals and cosmetics by RP-HPLC after pre-column derivatization, Farmaco, 1994, 49, 421-425. SAMPLE

Matrix: formulations Sample preparation: Ointment, lotion. Weigh out ointment or lotion equivalent to about 15 mg azelaic acid, dissolve in 100 mL MeOH, dilute an aliquot 1:5 with water. 200 jxL Sample + 150 |xL 20 mM tetrahexylammonium bromide in 100 mM pH 7.0 phosphate buffer + 100 |xL 4.2 mg/mL 2-bromoacetyl-6-methoxynaphthalene in acetone, stir for 33 min at 70, add 150 |JLL 20 |xg/mL IS in MeCN, sonicate for 1 min, inject a 50 |JLL aliquot into mobile phase A. Ointment. Dissolve in MeCN to give a concentration of 18 |xg/mL. 100-200 |xL Sample + 100 |xL 4.2 mg/mL 2-bromoacetyl-6-methoxynaphthalene in MeCN + 100 |xL 1% triethylamine in MeCN, heat at 40 for 40 min, reconstitute in 150 |xL 40 fjLg/mL IS in Mobile Phase B and 450 jxL mobile phase B, sonicate for 1 min, inject a 50 IxL aliquot into mobile phase B. Powder. Weigh out powder equivalent to about 15 mg azelaic acid, dissolve in 100 mL MeOH, sonicate for 10 min, centrifuge at 4000 rpm for 20 min, filter the supernatant, dilute an aliquot of the filtrate 1:5 with water. 200 |xL Sample + 150 |xL 20 mM tetrahexylammonium bromide in 100 mM pH 7.0 phosphate buffer + 100 |xL 4.2 mg/mL 2-bromoacetyl-6-methoxynaphthalene in acetone, stir for 33 min at 70, add 150 |xL 20 |xg/mL IS in MeCN, sonicate for 1 min, inject a 50 |xL aliquot into mobile phase A. (Synthesis of 2-bromoacetyl-6-methoxynaphthalene is as follows. Stir

equimolar amounts of 2-acetyl-6-methoxynaphthalene (6'-methoxy-2'-acetonaphthone, Aldrich) and methyltriphenylphosphonium tribromide in anhydrous THF under nitrogen at room temperature for 1 h, dilute the reaction mixture with ether, wash with sodium bisulfite solution, wash with water (Phosphorus and Sulfur 1985, 25, 357). [Bromination can also be achieved with phenyltrimethylammonium tribromide over 3 h but the reaction is less selective.] Purify the crude product by column chromatography on silica gel using chloroform:petroleum ether 50:50 to give 2-bromoacetyl-6-methoxynaphthalene (mp 109112) (Chromatographia 1992, 33, 13).)
HPLC VARIABLES

Column: 250 X 4.6 Hypersil 5 ODS Mobile phase: MeCN:MeOH:THF:water 38.5:28:3.5:30 (A) or 37.4:27.2:3.4:32 Column temperature: 35 Flow rate: 1.2 (A), 1.6 (B) Injection volume: 50 Detector: F ex 300 em 460
CHROMATOGRAM

Retention time: 18
Internal standard: valproic acid 6-methoxynaphthacylester (15.5) [Prepare by dissolving 2 mmoles valproic acid and 1 mmole 2-bromoacetyl-6-methoxynaphthalene in 10 mL anhydrous MeCN, add 0.5 mL triethylamine, heat at 60 for 30 min, cool, dilute with 30 mL water, extract three times with 10 mL portions of diethyl ether. Combine the extracts, wash with 5% sodium bicarbonate, wash three times with 10 mL portions of water, dry over anhydrous sodium sulfate, evaporate under reduced pressure, recrystallize from MeOH/water to give white crystals, mp 56-7 (Chromatographia 1992, 33, 13).]
KEY WORDS

ointment; lotion; powder; derivatization

REFERENCE Gatti,R.; Andrisano,V.; Di Pietra,A.M.; Cavrini,V. Analysis of aliphatic dicarboxylic acids in pharmaceuticals and cosmetics by liquid chromatography (HPLC) with fluorescence detection, J.Pharm.BiomedAnaL, 1995, 13, 589-595.

Azelastine
Molecular formula: C22H24CIN3O Molecular weight: 381.90 CAS Registry No.: 58581-89-8, 79307-93-0 (HCI) Merck Index: 939 Led nicer No.: 4 152

SAMPLE

Matrix: blood, tissue Sample preparation: 1 mL Plasma or tissue homogenate (equivalent to 100 mg tissue) + 100 IJLL 150 ng/mL IS + 250 JJLL 1 M NaOH + 9 mL hexaneroctanol 95:5, extract. Add the organic layer to 125 |xL 0.2% acetic acid, extract, centrifuge, inject 90 |xL of the aqueous layer.
HPLC VARIABLES

Column: 250 mm long 5 jjum Hypersil CPS Mobile phase: MeCN:9 mM pH 3.0 triethylammonium phosphate (sic) 50:50 (plasma) or MeCN:water:triethylamine:phosphoric acid 500:500:0.4:0.2 (tissue) Column temperature: 60 (tissue), 40 (plasma) Flow rate: 0.45 (plasma), 0.60 (tissue) Injection volume: 90 Detector: F ex 215 em 360
CHROMATOGRAM

Internal standard: 4 - (p-chlorobenzyl) - 2 - [N-methyl - 2,6 - ethanopiperidinyl - (4)] -1 - (2H) phthalazinone hydrochloride (9.97 (plasma), 9.61 (tissue)) Limit of quantitation: 36 ng/g (tissue), 0.288 ng/mL (plasma)
OTHER SUBSTANCES Extracted: metabolites KEYWORDS

plasma; guinea pig; lung

REFERENCE Adusumalli,V.E.; Wong,K.K.; Kucharczyk,N.; Sofia,R.D. Pharmacokinetics of azelastine and its active metabolite, desmethylazelastine, in guinea pigs, Drug Metab.Dispos., 1992, 20, 530-535. SAMPLE

Matrix: blood, tissue Sample preparation: Tissue. Homogenize lung tissue in ten volumes water (Tissumizer). 1 g Homogenate + 100 jxL 10 jxg/mL IS in 0.2% acetic acid + 100 yJL 10 M NaOH + 9 mL hexane:n-octanol 95:5, rotate for 1 h at 50 rpm, centrifuge. Remove 8 mL organic layer and add it to 125 |xL 0.2% acetic acid, vortex vigorously, inject an aliquot of the aqueous layer. Plasma. 1 mL Plasma + 100 JJLL 150 ng/mL IS in 0.2% acetic acid + 250 JiL 1 M NaOH + 9 mL hexanern-octanol 95:5, rotate for 1 h at 50 rpm, centrifuge. Remove 8 mL organic layer and add it to 125 |xL 0.2% acetic acid, vortex vigorously, inject an aliquot of the aqueous layer.
HPLC VARIABLES

Column: 250 X 2 5 |xm Hypersil CPS Mobile phase: MeCN:9 mM pH 3.0 triethylammonium phosphate (sic) 50:50 (plasma) or MeCN:water:triethylamine:phosphoric acid 500:500:0.4:0.2 (tissue)

Column temperature: 60 (tissue), 40 (plasma) Flow rate: 0.45 (plasma), 0.60 (tissue) Detector: F ex 215 em 360
CHROMATOGRAM

Retention time: 7.88 (plasma), 8.27 (tissue) Internal standard: 4 - (p - chlorobenzyl) - 2 - [N - methyl - 2,6 - ethanopiperidinyl - (4)] -1 - (2H) phthalazinone hydrochloride (9.97 (plasma), 9.61 (tissue)) Limit of quantitation: 0.039 ng/g (tissue), 0.156 ng/mL (plasma)
OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

plasma; guinea pig; lung

REFERENCE Langevin,C.N.; Pivonka,J.; Wichmann,J.K.; Kucharczyk,N.; Sofia,R.D. High performance liquid chromatographic determination of azelastine and desmethylazelastine in guinea pig plasma and lung tissue, Biomed.Chromatogr., 1993, 7, 7-11. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH and dilute with water to a concentration of 20 |xg/mL, inject a 10 jxL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 conalbumin-conjugated silica gel (React 2 g Unisil Q NH2 (MachereyNagel) and 3 g N,N-disuccinimidylcarbonate in 50 |xL MeCN for 6 h at room temperature, filter, wash silica gel with MeCN, wash with 50 mM pH 7.5 phosphate buffer. Add silica gel to 2 g conalbumin (ovotransferrin) (from egg white) in 50 mL 50 mM pH 7.5 phosphate buffer, stir at room temperature for 6 h, filter, wash with water, wash with isopropanol: water 1:2, pack into columns.) Mobile phase: EtOH:50 mM pH 5.0 potassium phosphate buffer 8:92
Flow rate: 1 Injection volume: 10 Detector: UV 230 CHROMATOGRAM Retention time: 20 (d), 26 (1)
KEYWORDS

chiral
REFERENCE Mano,N.; Oda,Y.; Miwa,T.; Asakawa,N.; Yoshida,Y.; Sato,T. Conalbumin-conjugated silica gel, a new chiral stationary phase for high-performance liquid chromatography, J.Chromatogr., 1992, 603, 105109. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 |xL aliquot of a solution in MeOH.

HPLC VARIABLES

Guard column: 4 X 4 5 |xm LiChrospher Si-60 Column: 250 X 4 5 |xm LiChrospher Si-60 Mobile phase: MeOH containing 0.033% perchloric acid

Flow rate: 0.5 for 17 min then 0.9 Injection volume: 20 Detector: F ex 210 em 360 CHROMATOGRAM Retention time: 27 Internal standard: azelastine OTHER SUBSTANCES Simultaneous: flezelastine KEYWORDS

azelastine is IS
REFERENCE Paris,S.; Blaschke,G.; Locher,M.; Borbe,H-O.; Engel,J. Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergenic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis, J.Chromatogr.B, 1997, 691, 463-471.

Azithromycin
Molecular formula: C38H72N2O12 Molecular weight: 749.00 CAS Registry No.: 83905-01-5 Merck Index: 946

SAMPLE

Matrix: blood Sample preparation: Mix 200 |L plasma with 50 |xL IS, add 200 JJLL 100 mM sodium carbonate, vortex for 30 s, add 3.5 mL MTBE, mix for 20 min, centrifuge at 2000 g for 10 min. Evaporate the MTBE layer to dryness at 37, reconstitute the residue in 200 |xL mobile phase, mix for 20 min, centrifuge, inject an 80 |xL aliquot.
HPLC VARIABLES

Column: 150 X 3.9 10 |xm ixBondapak C18 Mobile phase: MeCN:50 mM pH 7.5 phosphate buffer 55:45 Flow rate: 1 Injection volume: 80 Detector: E, ESA Coulochem 5100 A, ESA 5010 dual electrode analytical cell at +680 mV and +780 mV, ESA 5020 guard cell +1.0 V
CHROMATOGRAM

Internal standard: clarithromycin Limit of detection: 10 ng/mL

KEYWORDS

plasma; pharmacokinetics
REFERENCE Patel,K.B.; Xuan,D.; Tessier,P.R.; Russomanno,J.H.; Quintiliani,R.; Nightingale,C.H. Comparison of bronchopulmonary pharmacokinetics of clarithromycin and azithromycin, Antimicrob.Agents Chemother., 1996, 40, 2375-2379. SAMPLE

Matrix: blood, bronchoalveolar lavage fluid Sample preparation: Blood. Centrifuge 10 mL blood at 2500 g, separate the serum. Peripheral blood monocytes (PBM). Expose the PBM pellet to several freeze-thaw cycles, dilute the cell pellet in 1 mL saline with 1 mL serum and saline to volume 5 mL. Bronchoalveolar lavage (BAL) fluid. Expose the BAL pellet to several freeze-thaw cycles, dilute sample with water to volume 50 mL, freeze and thaw.
HPLC VARIABLES

Column: 50 X 4.6 5 jim Chromegabond Alkylphenyl aluminum oxide (E.S. Industries, N.J.) Mobile phase: MeCN:20 mM pH 10 Na3PO4 75:25 Flow rate: 1.5 Detector: E, Bioanalytical Systems, dual glassy carbon electrodes 600 and 850 mV, Ag/AgCl reference electrode
CHROMATOGRAM

Limit of detection: 2 ng/mL (BAL and leucocytes), 10 ng/mL (serum)

KEY WORDS

serum; pharmacokinetics
REFERENCE 01sen,K.M.; San Pedro,G.S.; Gann,L.R; Gubbins,P.O.; Halinski,D.M.; Campbell,G.D.,Jr. Intrapulmonary pharmacokinetics of azithromycin in healthy volunteers given five oral doses, Antimicrob.Agents Chemother., 1996, 40, 2582-2585. SAMPLE

Matrix: blood, gastric juice, gastric mucosa, saliva, vitreous humor Sample preparation: Homogenize 5-20 mg gastric mucosa in 300 jxL 10 mM pH 7.4 sodium phosphate buffer with sonication. Add 500 ng roxithromycin in MeOH:water 50:50 to 500 |xL plasma, serum, saliva, gastric juice, leucocytes lysate, vitreous humor, or 300 IxL gastric mucosa homogenate, vortex, add 200 jxL 100 mM sodium carbonate and 3 mL MTBE, shake thoroughly (5 X 2 s in an SMI Multi-tube vortexer), centrifuge at 1000 g for 5 min, freeze the aqueous layer in liquid nitrogen or in a freezer at -70 for 15 min. Evaporate the upper organic layer to dryness in a centrifugal vacuum evaporator (Jouan RC 10.22), reconstitute the residue in 250 |xL MeOH:water 50:50, inject a 20-50 JULL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 jxm Zorbax SB CN Mobile phase: MeCN:MeOH:50 mM Na2HPO4 and NaH2PO4 buffer 52.2:4.3:43.5 (pH 6.8) (The mobile phase was a mixture of 600 mL MeCN, 50 mL MeOH and 500 mL 50 mM Na2HPO4 and NaH2PO4 buffer.) Column temperature: 30 Flow rate: 1 Injection volume: 20-50 Detector: E, ESA Coulochem II, guard cell +1.0 V, screening cell E l +0.50 V, analytical cell E2 +0.80 V
CHROMATOGRAM

Retention time: 12 Internal standard: roxithromycin (5.5), clarithromycin (10) Limit of detection: 10 ng/mL
KEY WORDS

gastric juice; pharmacokinetics; plasma; saliva; serum

REFERENCE Kees,R; Spangler,S.; Wellenhofer,M. Determination of macrolides in biological matrices by high-performance liquid chromatography with electrochemical detection, J.Chromatogr.A, 1998, 812, 287-293. SAMPLE

Matrix: tears Sample preparation: Add 10 fiL 20 \L g/mL IS and 500 |xL of 60 mM sodium carbonate to 50 JULL tears. Extract with 5 mL of MTBE. Evaporate the organic layer to dryness under a stream of nitrogen at room temperature. Reconstitute the sample in 100 |xL MeCN: water 50:50, inject an 80 |xL aliquot.
HPLC VARIABLES

Guard column: 4 ^m Nova-Pak C18 Column: 100 X 8 4 |x m Nova-Pak C18 radial compression Mobile phase: MeCN:MeOH:buffer 19:9:75 (Buffer was 35 mM Na2HPO4 containing 5 mM tetrabutylammonium phosphate and 5 mM sodium perchlorate, adjusted to pH 7.0 with phosphoric acid. Mobile phase was recycled and the buffer was prepared fresh every other day.)

Column temperature: 26 Flow rate: 4.0 Injection volume: 80 Detector: E, Coulochem 11, 5021 conditioning cell 0.7 V, 5011 analytical cell at 0.85 V
CHROMATOGRAM

Internal standard: n-propylazithromycin Limit of quantitation: 100 ng/mL OTHER SUBSTANCES Extracted: metabolites
REFERENCE Raines,D.A.; Yusuf,A.; el-Yazigi,A. Simultaneous analysis of azithromycin and two of its metabolites in human tears (Abstract 2501), Pharm.Res., 1997, 14, S377-S377.

Azlocillin
Molecular formula: C20H23N5O6S Molecular weight: 461.50 CAS Registry No.: 37091-66-0, 37091-65-9 (monosodium salt) Merck Index: 947 Lednicer No.: 3 206

SAMPLE

Matrix: bile, blood, urine Sample preparation: Serum. 0.5 mL serum + 0.5 mL MeCN mix in 7 mL tube on vortex mixer; shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; transfer supernatant to another tube, add 7 aliquots dichloromethane; equilibrate 10 min; shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; inject aliquot of upper aqueous layer. Urine. Centrifuge urine and dilute 1:20. Bile. Centrifuge bile and dilute 1:10.
HPLC VARIABLES

Column: 75 X 4.6 3 |xm octadecylsilane Mobile phase: 20:80 MeCN:20 mM ammonium acetate adjusted to pH 5 with glacial acetic acid Flow rate: 1 Injection volume: 5 Detector: UV 214
CHROMATOGRAM

Retention time: 2.2 Limit of detection: 100 ng/mL

OTHER SUBSTANCES

Also analyzed: ampicillin, aztreonam, cefmenoxime, cefoperazone, cefsulodin, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, desacetylcefotaxime, mezlocillin, penicillin G, piperacillin, ticarcillin
KEY WORDS

serum
REFERENCE Jehl,R; Birckel,R; Monteil,H. Hospital routine analysis of penicillins, third-generation cephalosporins and aztreonam by conventional and high-speed high-performance liquid chromatography, J.Chromatogr., 1987, 413, 109-119. SAMPLE

Matrix: blood, bronchial secretions Sample preparation: Add an equal volume of digestor to the bronchial secretion, shake to fluidify sample. 1 mL Serum or diluted bronchial secretion + 1 mL MeCN, vortex, centrifuge at 5000 rpm for 10 min. Remove the supernatant and add it to 4 mL dichloromethane, vortex, centrifuge. Remove the aqueous supernatant and inject an aliquot.
HPLC VARIABLES

Column: endcapped 5 jxm Lichrospher RP 18 Mobile phase: MeCN:pH 7 phosphate buffer 20:80 Flow rate: 0.8 Injection volume: 4.10 Detector: UV 220

CHROMATOGRAM
Retention time: 20

KEYWORDS

serum
REFERENCE Condomines,M.; Mallet,M.N.; Albanese,J.; Gouin,R; De Micco,R A rapid high-performance liquid chromatography method for determining p-lactam antibiotics in biological fluids and tissues, Chemioterapia, 1987, 6, 251-253. SAMPLE

Matrix: blood, urine Sample preparation: Dilute urine 1:20 with water. 200 JULL Serum or diluted urine + 1 mL 20 jxg/mL mezlocillin in MeCN, vortex, centrifuge, inject a 10-20 |xL aliquot of the supernatant.
HPLC VARIABLES

Column: 250 X 4.6 5 fxm Ultrasphere C18 Mobile phase: MeOH:67 mM pH 3.0 KH2PO4 45:55 Flow rate: 1 Injection volume: 10-20 Detector: UV 231.1
CHROMATOGRAM

Retention time: 10.1 Internal standard: mezlocillin (12.9)

KEY WORDS

serum; pharmacokinetics
REFERENCE Barriere,S.L.; Catlin,D.H.; Orlando,P.L.; Noe,A.; Frost,R-\V. Alteration in the pharmacokinetic disposition of ciprofloxacin by simultaneous administration of azlocillin, Antimicrob.Agents Chemother., 1990, 34, 823-826.

Azosemide
Molecular formula: C12H11CIN6O2S2 Molecular weight: 370.84 CAS Registry No.: 27589-33-9 Merck Index: 953 Lednicer No.: 3 27 SAMPLE

Matrix: blood, tissue, urine Sample preparation: Plasma, urine. 50 |xL Plasma or urine + 125 JJLL MeCN, vortex, centrifuge at 9000 g for 10 min, inject 50 (JLL of the supernatant. Tissue. Homogenize with four volumes of 0.9% NaCl (Tissuemizer), centrifuge at 9000 g for 10 min. Remove 50 JULL of the supernatant and add it to 125 |xL MeCN, vortex, centrifuge at 9000 g for 10 min, inject a 50 (xL aliquot of the supernatant.
HPLC VARIABLES

Column: 300 X 3.9 10 jjim C18 (Waters) Mobile phase: MeCN:30 mM phosphoric acid 40:50 Flow rate: 1.5 Injection volume: 50 Detector: UV 240
CHROMATOGRAM

Retention time: 6.0 Limit of detection: 50 ng/mL

OTHER SUBSTANCES

Extracted: metabolites Noninterfering: furosemide, bumetanide, hydrochlorothiazide, amiloride, spironolactone

KEYWORDS

plasma; human; rabbit; rat; blood; liver; lung; heart; brain; kidney; muscle; stomach; intestine; spleen; pharmacokinetics
REFERENCE Lee,S.H.; Lee,M.G. Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography, J.Chromatogr.B, 1994, 656, 367-372. SAMPLE

Matrix: blood, urine Sample preparation: Urine. Filter (0.45 |xm). Remove a 300 JJIL aliquot and add it to 300 IxL water and 50 |xL 412 |xg/mL phenobarbital in EtOH, vortex, inject a 10 |JLL aliquot. Serum. 300 uL (?) Serum + 20 JJLL 412 jjig/mL phenobarbital in EtOH + 400 uL MeCN, mix, centrifuge. Remove the supernatant and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 50-100 jxL buffer, inject a 5-20 |xL aliquot.
HPLC VARIABLES

Guard column: 70 X 2.1 CO:Pell ODS (Whatman) Column: 250 X 4.6 5 |xm Zorbax ODS C18 Mobile phase: Gradient. MeCN:buffer from 10:90 to 40:60 over 10 min, maintain at 40:60 for 2 min, re-equilibrate for 5 min. (Buffer was 0.6 mL glacial acetic acid in 1 L water, adjust pH to 4.05 with 4 M NaOH.) Flow rate: 2 Injection volume: 5-20

Detector: UV 239
CHROMATOGRAM

Retention time: 10 Internal standard: phenobarbital (11) Limit of detection: 50 ng/mL

OTHER SUBSTANCES

Noninterfering: acetaminophen, aspirin, chlorothiazide, chlorpromazine, hydrochlorothiazide, procainamide, quinidine, sulfamethoxazole, theophylline, tolbutamide
KEY WORDS

serum; pharmacokinetics
REFERENCE Seiwell,R.; Brater,C. Separation and analysis of azosemide in urine and in serum by high-performance liquid chromatography, J.Chromatogr., 1980, 182, 257-261. SAMPLE

Matrix: blood, urine Sample preparation: 100 J U L L Plasma or urine + 250 jxL MeCN, vortex, centrifuge, inject an aliquot of the supernatant.
HPLC VARIABLES

Column: 300 X 3.9 10 um C18 (Waters) Mobile phase: MeCN:30 mM phosphoric acid 40:50 Flow rate: 1.5 Detector: UV 240
CHROMATOGRAM

Retention time: 6 Limit of detection: 90 ng/mL

KEYWORDS

rabbit; plasma; pharmacokinetics

REFERENCE Lee,S.H.; Shin,W.G.; Lee,M.G.; Kim,N.D. Arterial and venous blood sampling in pharmacokinetic studies: azosemide in rabbits, Biopharm.Drug Dispos., 1994, 25, 305-316.

Aztreonam
Molecular formula: C13H17N5O8S2 Molecular weight: 435.44 CAS Registry No.: 78110-38-0 Merck Index: 955 Led nicer No.: 4 193, 195

SAMPLE

Matrix: bile, blood, urine Sample preparation: Serum. 0.5 mL serum + 0.5 mL MeCN mix in 7 mL tube on vortex mixer; shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; transfer supernatant to another tube, add 7 aliquots dichloromethane; equilibrate 10 min; shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; inject aliquot of upper aqueous layer. Urine. Centrifuge urine and dilute 1:20. Bile. Centrifuge bile and dilute 1:10.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Ultrasphere ODS Mobile phase: 33:67 MeCN: 10 mM ammonium acetate + 5 mM tetrabutylammonium bromide adjusted to pH 7 with glacial acetic acid Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 4.2 Limit of detection: 500 ng/mL

OTHER SUBSTANCES

Also analyzed: ampicillin, azlocillin, cefmenoxime, cefoperazone, cefsulodin, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, desacetylcefotaxime, mezlocillin, penicillin G, piperacillin, ticarcillin
KEYWORDS

serum
REFERENCE Jehl,R; Birckel,R; Monteil,H. Hospital routine analysis of penicillins, third-generation cephalosporins and aztreonam by conventional and high-speed high-performance liquid chromatography, J.Chromatogr., 1987, 413, 109-119. SAMPLE

Matrix: blood, urine Sample preparation: Serum. Dilute serum with an equal volume of MeCN, centrifuge at 15000 g for 2 min, inject a 50-200 jxL aliquot of the supernatant. Urine. Dilute urine tenfold with 5 mM pH 3.0 tetrabutylammonium hydrogen sulfate, inject a 50-200 uL aliquot.
HPLC VARIABLES

Guard column: 30 X 3.9 Bondapak C18/Corasil Column: 300 x 3.9 jxBondapak C18 Mobile phase: Human serum, human urine. MeCN:5 mM tetrabutylammonium hydrogen sulfate and 5 mM ammonium sulfate adjusted to pH 3.0 with 1 M K2HPO4 20:80; Mouse serum, mouse urine, monkey urine. MeCNrbuffer 20:80; Rat serum, monkey serum. MeCN:buffer 35:65; Rat urine, rabbit urine. MeCN:buffer 17.5:82.5; Rabbit serum. MeCN:

buffer 25:75 (Buffer was 5 mM tetrabutylammonium hydrogen sulfate adjusted to pH 3.0 with 1 M K2HPO4.) Flow rate: 2 Injection volume: 50-200 Detector: UV 293
CHROMATOGRAM

Retention time: 5 (human serum and urine) Limit of detection: 5000 ng/mL (urine), 1000 ng/mL (serum)
KEYWORDS

serum; human; monkey; rat; mouse; rabbit

REFERENCE Pilkiewicz,F.G.; Remsburg,B.J.; Fisher,S.M.; Sykes,R.B. High-pressure liquid chromatographic analysis of aztreonam in sera and urine, Antimicrob.Agents Chemother., 1983, 23, 852-856. SAMPLE

Matrix: formulations Sample preparation: 100 (xL Solution + 4.9 mL MeOH:water 20:80, inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 5 fim Adsorbosphere C18 Column: 250 X 4.6 5 (xm Adsorbosphere C18 Mobile phase: MeCN:5 mM pH 2.6 ammonium phosphate containing 2 mM tetrabutylammonium hydroxide 26:74 Flow rate: 1 Injection volume: 50 Detector: UV 238 CHROMATOGRAM Retention time: 14.0
KEYWORDS

stability-indicating; injections; saline

REFERENCE Inagaki,K.; Gill,M.A.; Okamoto,M.R; Takagi,J. Stability of ranitidine hydrochloride with aztreonam, ceftazidime, or piperacillin sodium during simulated Y-site administration, Am. J.Hosp.Pharm., 1992, 49, 2769-2772. SAMPLE

Matrix: formulations Sample preparation: Dilute 1: 8 with water, combine a 100 |xL aliquot of the diluted solution with 100 |xL cimetidine solution and 200 |xL water, inject a 20 (xL aliquot.
HPLC VARIABLES

Column: 3.9 X 300 ixBondapak C18 Mobile phase: 5 mM tetrabutylammonium hydrogen sulfate, 7% MeCN, 14% MeOH in 10 mM phosphate buffer (pH 2.6-2.7) Flow rate: 1 Injection volume: 20 Detector: UV 225
CHROMATOGRAM

Retention time: 7.05 Internal standard: cimetidine (3.27)

OTHERSUBSTANCES

Simultaneous: with ampicillin, sulbactam

KEY WORDS

injections; stability-indicating; saline

REFERENCE Belliveau,P.R; Nightingale,C.H.; Quintiliani,R- Stability of aztreonam and ampicillin sodium-sulbactam sodium in 0.9% sodium chloride injection, Am.J.Hosp.Pharm., 1994, 52, 901-904. SAMPLE

Matrix: formulations Sample preparation: Add cefazolin sodium (20 mg/mL), inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 fiBondapak C18 Mobile phase: MeCN:buffer 20:80 (Buffer was 5 mM tetrabutylammonium hydrogen sulfate, adjusted to pH 3 with 1 M KH2PO4.) Flow rate: 2 Injection volume: 10 Detector: UV 293
CHROMATOGRAM

Retention time: 7.5 Internal standard: cefazolin (13)

KEYWORDS

injections; stability-indicating; 5% dextrose

REFERENCE Bosso,J.A.; Prince,R-A.; Fox,J.L. Compatibility of ondansetron hydrochloride with fluconazole, ceftazidime, aztreonam, and cefazolin sodium under simulated Y-site conditions, Am. J.Hosp.Pharm., 1994, 51, 389-391. SAMPLE

Matrix: formulations Sample preparation: Dilute with mobile phase, inject a 15 u.L aliquot.
HPLC VARIABLES

Guard column: 5 |xm C8 (Vydac) Column: 250 X 4.6 5 |xm C8 (Vydac) Mobile phase: MeOH:buffer 15:85 (Buffer was 50 mM KH2PO4 adjusted to pH 3.0 with phosphoric acid.) Flow rate: 1 Injection volume: 15 Detector: UV 270 CHROMATOGRAM Retention time: 7.8 OTHER SUBSTANCES Simultaneous: vancomycin
KEY WORDS

stability-indicating; injections; 5% dextrose; saline

REFERENCE Trissel,L.A.; Xu,Q.A.; Martinez,J.F. Compatibility and stability of aztreonam and vancomycin hydrochloride, Am. J.Health-Syst.Pharm., 1995, 52, 2560-2564. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 6jjim Zorbax CN Mobile phase: 1.8 mM pH 4.1 copper sulphate Flow rate: 0.7 Injection volume: 25 Detector: UV 232
CHROMATOGRAM

Retention time: 10.3 Limit of quantitation: 50 ng/mL OTHER SUBSTANCES Simultaneous: arginine
REFERENCE Khedr,A. High-performance liquid chromatography of ct-amino acids and aztreonam on reversed phase columns with aqueous Cu2+ as eluent, Biomed.Chromatogr., 1996, 10, 167-171. SAMPLE

Matrix: solutions Sample preparation: Add 20 |JLL solution to 2 mL 1 mg/mL cefoperazone in water, vortex for 15 s, inject a 10 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 10 jim Alltech C8 Mobile phase: MeCNrbuffer 23:77 containing 1.7 g/L tetrabutylammonium hydrogen sulfate, pH adjusted to 3.5 with 5 M NaOH (The buffer was 60 mL 100 mM sodium acetate and 710 mL 100 mM acetic acid.) Flow rate: 2 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 8.0 Internal standard: cefoperazone (13.5)

OTHER SUBSTANCES

Simultaneous: degradation products

KEYWORDS

5% dextrose; saline
REFERENCE Marble,D.A.; Bosso,J.A.; Townsend,R.J. Compatibility of clindamycin phosphate with aztreonam in polypropylene syringes and with cefoperazone sodium, cefonicid sodium, and cefuroxime sodium in partial-fill glass bottles, Drug Intell.Clin.Pharm., 1988, 22, 54-57.

Bacampicillin
Molecular formula: C21H27N3O7S Molecular weight: 465.53 CAS Registry No.: 50972-17-3, 37661-08-8 (HCI) Merck Index: 961 Lednicer No.: 3 204

SAMPLE

Matrix: blood Sample preparation: Add 100000 fold excess of ampicillin to eliminate adsorption of bacampicillin, extract into butyl acetate, re-extract into pH 2 buffer. Wash the aqueous phase with n-hexane, inject an aliquot.
HPLC VARIABLES

Column: 100 X 2.9 5 [xm Nucleosil C18 Mobile phase: MeCN:pH 7.4 phosphate buffer 41:49 Injection volume: 20 Detector: F with post-column reaction. The column effluent mixed with two volumes of sodium borate buffer then with one volume of 150 p,g/mL fluorescamine in acetone (Science 1972, 178, 871).
CHROMATOGRAM

Limit of detection: 800 pg/mL

REFERENCE Sjovall,J.; Westerlund,D.; Alvan,G.; Magni,L.; Nord,C.E.; Sorstad,J. Rectal bioavailability of bacampicillin hydrochloride in man as determined by reversed-phase liquid chromatography, Chemotherapy, 1984, 30, 137-147. SAMPLE Matrix: blood

Sample preparation: 0.5 mL Plasma + 1 mL MeOH, stir for 5 min, centrifuge at 2400 g for 10 min. Remove 1 mL supernatant, add 2 |xg cefazolin, inject an aliquot.
HPLC VARIABLES

Column: 300 X 3.9 5 |xm jjiBondapak C18 Mobile phase: MeOH:67 mM KH2PO4 20:80 Flow rate: 1.5 Injection volume: 50 Detector: UV 225
CHROMATOGRAM

Retention time: 9 (measured as ampicillin peak) Internal standard: cefazolin (14) Limit of detection: 500 ng/mL
KEYWORDS

plasma
REFERENCE Marzo,A.; Monti,N.; Ripamonti,M.; Arrigoni Martelli,E.; Picari,M. High-performance liquid chromatographic assay of ampicillin and its prodrug lenampicillin, J.Chromatogr., 1990, 507, 235-239.

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 14.687
KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163.

Bacitracin
Molecular formula: C66H103N17O16S (bacitracin A) Molecular weight: 1422.61 (bacitracin A)

CAS Registry No.: 1405-87-4, 1405-89-6 (zinc salt), 1405-88-5 (methylenedisalicylate) Merck Index: 965

SAMPLE Matrix: bulk

Sample preparation: Dissolve 1 g in 100 mL 20 mM HCl in MeOH:water 80:20, mix, vortex, centrifuge, filter (0.45 Jim), inject a 100 JJLL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm YMC basic C8 200 A (YMC) Mobile phase: Gradient. MeOH:50 mM pH 6.5 KH2PO4 from 57:43 to 63:37 over 1 h Column temperature: 25 Flow rate: 1 Injection volume: 100 Detector: UV 215
CHROMATOGRAM

Retention time: 14, 16, 20, 21, 25, 27, 30 (bioactive fractions)
REFERENCE Bell,R.G. Preparative high-performance liquid chromatographic separation and isolation of bacitracin components and their relationship to microbiological activity, J.Chromatogr., 1992, 590, 163-168. SAMPLE

Matrix: solutions Sample preparation: Dissolve in saline at a concentration of 1-10 mg/mL, inject an aliquot.
HPLC VARIABLES

Column: 300 X 3.9 C18 (Vydac) Mobile phase: Gradient. MeCN:water (both containing 0.05% trifluoroacetic acid) from 5: 95 to 65:35 over 20 min Flow rate: 2 Detector: UV 210
KEYWORDS

saline

REFERENCE Drapeau,G.; Petitclerc,E.; Toulouse,A.; Marceau,F. Dissociation of the antimicrobial activity of bacitracin USP from its renovascular effects, Antimicrob.Agents Chemother., 1992, 36, 955-961.

Baclofen
Molecular formula: C10H12CINO2 Molecular weight: 213.66 CAS Registry No.: 1134-47-0 Merck Index: 967 Lednicer No.: 2 121

SAMPLE

Matrix: CSF Sample preparation: Prepare a 100 X 7 column of Dowex 50X4-400, treat with an excess of aqueous ammonia, wash with water until the washings are neutral, wash with an excess of 4 M HCl, wash with water until the washings are neutral, rinse with 8 mL water. Add 200 jxL CSF to the column, wash with 8 mL water, elute with 2 mL 10% ammonia. Lyophilize the eluate, reconstitute the residue with 1 mL pH 7.4 phosphate buffer, extract twice with 1 mL portions of 1-butanol. Combine the organic layers and evaporate them to dryness, add 10-50 uL EtOH:water:triethylamine 50:25:25, evaporate to dryness, add 20 fxL reagent, let stand at room temperature for 20 min, evaporate to dryness, reconstitute with pH 7.4 sodium phosphate buffer, inject a 5-25 |xL aliquot. (Reagent was EtOH:triethylamine:water:phenylisothiocyanate 70:10:10:10, store at -20 (Anal.Biochem. 1989, 176, 269).)
HPLC VARIABLES

Column: 150 X 3.9 Pico-Tag Mobile phase: Gradient. A was sodium acetate adjusted to pH 6.4 with glacial acetic acid. B was MeCN:water 60:40. A:B from 100:0 to 60:40 over 10 min, to 0:100 over 1 min, maintain at 0:100 over 3 min. Column temperature: 38 Injection volume: 5-25 Detector: UV 254
CHROMATOGRAM

Retention time: 13.2 Limit of detection: 5-10 ng/mL

KEY WORDS

derivatization; SPE; pharmacokinetics

REFERENCE Sallerin-Caute,B-*> Monsarrat,B.; Lazorthes,Y.; Cros,J.; Bastide,R. A sensitive method for the determination of baclofen in human CSF by high performance liquid chromatography, J.Liq.Chromatogr., 1988, H1 1753-1761. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL Bond Elut SCX strong cation exchange SPE cartridge with 2 mL hexane, 2 mL MeOH, 2 mL water, and 3 mL saturated NaCl solution. 1 mL Plasma + 100 u.L water + 1 mL citrate buffer, mix, add to the SPE cartridge, wash with 4 mL water, wash with 1 mL saturated NaCl solution, dry the SPE cartridge, elute with 1.5 mL pH 10.4 borate buffer. Mix a 200 fxL aliquot of the eluate and add it to 50 IxL reagent, mix, inject a 20 |xL aliquot. (Prepare citrate buffer by diluting 89 mL 100 mM citric acid to 100 mL with 200 mM Na2HPO4, pH 2.6. Prepare pH 10.4 borate buffer by mixing 54 mL 200 mM boric acid in 100 mM NaOH with 46 mL 100 mM NaOH. Prepare pH 9.3 borate buffer by mixing 87 mL 200 mM boric acid in 100 mM NaOH with

13 mL 100 mM NaOH. Prepare reagent daily by mixing 75 mg o-phthalaldehyde, 5 mL MeOH, 50 |xL tert-butanethiol, and 5 mL pH 9.3 borate buffer.)
HPLC VARIABLES

Column: 150 X 3.9 4 jxm Novapak Mobile phase: MeOH:buffer 74:36 (Prepare buffer by adjusting the pH of 60 mM Na2HPO4 to 7 with 60 mM KH2PO4.) Flow rate: 0.8 Injection volume: 20 Detector: E, ESA Coulochem II, Model 5020 guard cell +1.2 V, Model 5011 glassy carbon working cell, screen electrode +0.2 V, quantifying electrode +0.7 V
CHROMATOGRAM

Retention time: 26 Limit of detection: 2.5 ng/mL Limit of quantitation: 10 ng/mL

KEYWORDS

derivatization; plasma; SPE; pharmacokinetics

REFERENCE Millerioux,L.; Brault,M.; Gualano,V.; Mignot,A. High-performance liquid chromatographic determination of baclofen in human plasma, J.Chromatogr.A, 1996, 729, 309-314. SAMPLE

Matrix: blood, CSF, dialysate, tissue Sample preparation: Dilute dialysate and CSF. Homogenize brain tissue with 4 volumes of MeOHrwater in an ice bath, let stand at -20 for 8 h. 30 \LL Plasma + 60 |JLL MeOH, mix, let stand at -20 for 1 h, centrifuge at 4 at 10000 rpm for 5 min, dilute the supernatant with water. Mix an aliquot of tissue homogenate, deproteinized plasma, diluted dialysate, or diluted CSF with an equal volume of the reagent, let stand for 1.5 min, inject a 30 |xL aliquot. (Prepare reagent by mixing 50 mg o-phthalaldehyde, 900 |xL MeOH, 100 fiL 400 mM pH 9.2 borate buffer, and 50 jxL 2-mercaptoethanol.)
HPLC VARIABLES

Guard column: ixBondapak C18 Guard-Pak Column: 250 X 4.6 5 jim Finepak SIL C18S ODS (Jasco) Mobile phase: MeOH:THF:100 mM pH 6.95 acetate buffer 45.5:2:52.5 (dialysate, CSF, plasma) or 43:2:55 (tissue) Flow rate: 1 Injection volume: 30 Detector: F ex 368 em 434
CHROMATOGRAM

Limit of detection: 100 nM

KEYWORDS

plasma; derivatization; rat; brain; pharmacokinetics

REFERENCE Deguchi,Y; Inabe,K.; Tomiyasu,K.; Nozawa,K.; Yamada,S.; Kimura,R. Study on brain interstitial fluid distribution and blood-brain barrier transport of baclofen in rats by microdialysis, Pharm.Res., 1995, 12, 1838-1844. SAMPLE

Matrix: blood, urine

Sample preparation: 500 |xL Plasma + 2 mL MeOH, centrifuge. 1 mL Supernatant or 100 J U L L urine + 500 jxL pH 9 sodium tetraborate buffer + 250 JJLL 0.2% 4-chloro-7-nitrobenzofurazan in MeOH, heat at 60 for 45 min, acidify with 100 mM HCl, extract with 5 mL ethyl acetate. Evaporate 3 mL of the extract to about 500 jiL, dry with anhydrous sodium sulfate, pass through SPE column, elute with ethyl acetate to give a final volume of 2 mL. Evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 1 mL mobile phase, inject a 10 U | LL aliquot. (Prepare the 25 X 6 SPE column with 0.0630.200 mm silica gel (Merck), wet it with ethyl acetate.)
HPLCVARIABLES

Column: 300 X 3.9 10 jxm Bondapak C18 Mobile phase: MeOH:water 45:55 Flow rate: 0.4 Injection volume: 10 Detector: F ex 463 em 524
CHROMATOGRAM

Retention time: 4.0 Limit of detection: 100 ng/mL (urine), 20 ng/mL (plasma)
KEYWORDS

plasma; pharmacokinetics; SPE; derivatization

REFERENCE Tosunoglu,S.; Ersoy,L. Determination of baclofen in human plasma and urine by high-performance liquid chromatography with fluorescence detection, Analyst, 1995, 120, 373-375. SAMPLE

Matrix: formulations Sample preparation: Dilute an aliquot with water to a concentration of 5 (xg/mL, filter (0.22 |xm), inject a 15 u,L aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 ^m Speri-5 ODS (Applied Biosystems) Mobile phase: MeCN:50 mM NaH2PO4 20:80, adjusted to pH 3.5 with 85% phosphoric acid Flow rate: 1 Injection volume: 15 Detector: UV 220
CHROMATOGRAM Retention time: 5.5 KEYWORDS

syrup; stability-indicating
REFERENCE Johnson,C.E.; Hart,S.M. Stability of an extemporaneously compounded baclofen oral liquid, Am.J.Hosp.Pharm., 1993, 50, 2353-2355. SAMPLE

Matrix: formulations Sample preparation: Dilute formulation 1:10 with water, inject a 50 |xL aliquot.
HPLC VARIABLES Guard column: 5 x 4 35-60 ^m Perisorb RP18 Column: 250 X 4 10 fjim LiChrosorb RP18 Mobile phase: MeOH:MeCN:2.72 g/L KH 2 PO 4 2:12:86

Injection volume: 50 Detector: UV 220 CHROMATOGRAM Retention time: 10.3

KEY WORDS

injections; water
REFERENCE Sadjak,A.; Wintersteiger,R- Compatibility of morphine, baclofen, floxuridine and fluorouracil in an implantable medication pump, Arzneimittelforschung, 1995, 45, 93-98. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 5 jxm Adsorbosphere C18 Mobile phase: MeOH.buffer 40:60 (Buffer was 50 mM sodium acetate adjusted to pH 6.4 with glacial acetic acid.) Flow rate: 1.0 Injection volume: 100 Detector: UV 266
CHROMATOGRAM

Limit of quantitation: 10 fxg/mL

REFERENCE A simple, rapid and reliable HPLC method for the analysis of baclofen in tablets (Abstract 3350), Pharm.Res., 1997, 14, S581-S581. SAMPLE

Matrix: solutions Sample preparation: Mix sample:50 (?) mM NaCN in 50 mM pH 9.3 borate buffer: 25 (?) mM naphthalene-2,3-dicarboxaldehyde in MeOH 3:1:1, let stand for 15 min, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 200 X 3 5 |xm Chromspher ODS-2 C18 (Chrompack) Mobile phase: Gradient. A was THF:50 mM pH 6.8 potassium phosphate buffer 5:95. B was MeCN:MeOH:50 mM pH 6.8 potassium phosphate buffer 55:10:35. A:B from 70:30 to 0:100 over 1 h, maintain at 0:100 for 20 min. Flow rate: 0.5 Injection volume: 50 Detector: F ex 420 CHROMATOGRAM Retention time: 32
OTHER SUBSTANCES

Simultaneous: amphetamine, tranylcypromine

KEY WORDS

derivatization

REFERENCE Koning,H.; Wolf,H-; Venema,K; Korf,J. Automated precolumn derivatization of amino acids, small peptides, brain amines and drugs with primary amino groups for reversed-phase high-performance liquid chromatography using naphthalenedialdehyde as the fluorogenic label, J.Chromatogr., 1990, 533, 171-178. SAMPLE

Matrix: solutions Sample preparation: 200 fiL 10 mM Baclofen in 100 mM sodium bicarbonate + 200 |xL 10 mM l-fluoro-2,4-dinitrophenyl-5-L-alanine amide in acetone (freshly prepared), stir at 40 for 1 h, cool, add 100 JJLL 200 mM HCl, inject an aliquot. (Derivatization in Anal.Biochem. 1992, 202, 210.)
HPLC VARIABLES

Column: 150 X 4.6 5 ^m YMCGEL C8 (YMC) Mobile phase: MeOH:5% KH2PO4 (pH 4.0) 11:8 Flow rate: 1 Detector: UV 340
CHROMATOGRAM

Retention time: 17 (R-(-)), 20 (S-(+))

KEYWORDS

chiral; derivatization
REFERENCE Shimada,K; Mitamura,K.; Morita,M.; Hirakata,K Separation of the diastereomers of baclofen by high performance liquid chromatography using cyclodextrin as a mobile phase additive, J.Liq. Chromatogr., 1993, 16, 3311-3320. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 |mm LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acidrbuffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 5.71 (A), 3.47 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, fu-

rosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119. SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a 100 fxg/mL solution in mobile phase.
HPLC VARIABLES

Column: 150 X 4 5 |xm Crownpak CR(+) immobilized crown ether Mobile phase: MeOH:0.1% pH 1.9 perchloric acid 15:85 Column temperature: 40 Flow rate: 1 Detector: UV 210
CHROMATOGRAM Retention time: 9.32, 14.23 OTHER SUBSTANCES

Simultaneous: levodopa, norephedrine, primaquine

KEYWORDS

chiral; comparison with capillary electrophoresis

REFERENCE Nishi,H.; Nakamura,K.; Nakai,H.; Sato,T. Separation of enantiomers and isomers of amino compounds by capillary electrophoresis and high-performance liquid chromatography utilizing crown ethers, J.ChromatogrA, 1997, 757, 225-235.

Bambuterol
Molecular formula: C18H29N3O5 Molecular weight: 367.45

CAS Registry No.: 81732-65-2, 81732-46-9 (HCI) Merck Index: 980 SAMPLE Matrix: blood

Sample preparation: Dilute plasma with buffer containing IS, inject an aliquot corresponding to 0.5 |xL plasma.
HPLC VARIABLES

Guard column: 4 x 4 LiChrosorb RP-select B Column: 50 X 4 5 jxm LiChrospher RP-select B Mobile phase: Gradient. MeOH: 100 mM pH 5 ammonium acetate from 3:97 to 10:90 over 5 min, to 25:75 over 1 min, to 38:62 over 7 min Flow rate: 1.4 Detector: MS, thermospray, Finnigan 4500 quadrupole, ion source repeller equipped with polyimide sleeve, trap at -90, analyzer pressure 0.000035 Tbrr, manifold fore pressure 0.20 Torr, exhaust line pressure 1.1 Torr, repeller 45 V, vaporizer 115, jet 200, aerosol 270
CHROMATOGRAM

Retention time: 11 Internal standard: hedxadeuterobambuterol

KEYWORDS

plasma; LC-MS; dog

REFERENCE Lindberg,C; Paulson, J.; Blomqvist,A. Evaluation of an automated thermospray liquid chromatographymass spectrometry system for quantitative use in bioanalytical chemistry, J.Chromatogr., 1991,554, 215-226. SAMPLE

Matrix: microsomal incubations Sample preparation: Condition a C18 Sep-Pak SPE cartridge with three 3 mL portions of EtOH, two 3 mL portions of water, and with 3 mL 10 mM pH 7.5 phosphate buffer. Add the incubation mixture to the SPE cartridge, wash with two 3 mL portions of water, elute with two 1 mL portions of EtOH:50 mM pH 8.5 ammonium chloride buffer 95:5. Evaporate the eluate to dryness under a stream of nitrogen at 60, reconstitute the residue in 500 JJLL of the initial mobile phase, inject a 200 |xL aliquot.; SPE
HPLC VARIABLES Column: 150 X 5 Nucleosil 10SA

Mobile phase: Gradient. A was 250 mM pH 4.6 ammonium acetate buffer. B was MeCN: 500 mM pH 4.6 ammonium acetate buffer 50:50. From A:B 90:10 to 10:90 over 20 min. Flow rate: 1 Injection volume: 200 Detector: UV 254
CHROMATOGRAM Retention time: 16.5

OTHER SUBSTANCES

Extracted: terbutaline, metabolites

KEYWORDS

rat; SPE
REFERENCE Lindberg,C; Roos,C; Tunek,A.; Svensson,L.A. Metabolism of bambuterol in rat liver microsomes: identification of hydroxylated and demethylated products by liquid chromatography mass spectrometry, Drug Metab.Dispos., 1989, 17, 311-322. SAMPLE

Matrix: perfusate, tissue Sample preparation: Perfusate. 400 fiL Lung perfusate + 400 |xL 5% perchloric acid, mix, centrifuge, inject a 200 |xL aliquot of the supernatant. Tissue. Homogenize lung in 2 volumes of water (Polytron Homogenizer), mix 400 |xL homogenate with 400 |xL 5% perchloric acid, mix, centrifuge, inject a 200 jxL aliquot.
HPLC VARIABLES

Column: 150 X 5 Nucleosil 10SA Mobile phase: Gradient. A was 19.3 g ammonium acetate and 14.4 mL acetic acid in 1 L water. B was 19.3 g ammonium acetate and 14.4 mL acetic acid in 1 L MeCN:water 50: 50. A:B from 90:10 to 10:90 over 20 min, stay at 10:90 for 3 min, return to initial conditions over 3 min, re-equilibrate for 7 min. Flow rate: 1 Injection volume: 200 Detector: UV 254
CHROMATOGRAM Retention time: 18 OTHER SUBSTANCES

Extracted: metabolites, terbutaline

KEY WORDS

lung; guinea pig

REFERENCE Ryrfeldt,A.; Nilsson,E.; Tunek,A.; Svensson,L.-A. Bambuterol: uptake and metabolism in guinea pig isolated lungs, Pharm.Res., 1988, 5, 151-155.

Bamethan
Molecular formula: C12H19NO2 Molecular weight: 209.29 CAS Registry No.: 3703-79-5,5716-20-1 (sulfate) Merck Index: 981 Led nicer No.: 2 39 SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |ULL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 (Jim Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 5.91 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 jig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7 Flow rate: 2 Injection volume: 20 Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V

CHROMATOGRAM Retention time: 1.7 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, metnamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopr amide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. IL Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Bamifylline
Molecular formula: C20H27N5O3 Molecular weight: 385.47 CAS Registry No.: 2016-63-9, 20684-06-4 (HCI) Merck Index: 982 Lednicer No.: 1 426

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 JULL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jiL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 fim Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 207.5 CHROMATOGRAM Retention time: 10.288 KEYWORDS whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Barbital
Molecular formula: C8H12N2O3 Molecular weight: 184.19 CAS Registry No.: 57-44-3, 144-02-5 (sodium salt) Merck Index: 989 LednicerNo.: 1 267 SAMPLE M a t r i x : blood

Sample preparation: Inject a 5-20 mobile phase A or B.

HPLC VARIABLES

JJLL

aliquot of serum directly onto the column with

Column: 100 X 4.6 5-10 (xm Silicalite (by sieving Silicalite, 3M Co.(?)) Mobile phase: MeCN:20 mM pH 6.9 phosphate buffer 8:92 (A) or Gradient. MeCN:20 mM pH 6.9 phosphate buffer from 5:95 to 20:80 over 2 min, to 25:75 over 2 min, to 30:70 over 4 min, to 50:50 over 2 min, maintain at 50:50 for 10 min (B) Flow rate: 1 Injection volume: 5 (A), 20 (B) Detector: UV 254
CHROMATOGRAM

Retention time: 7.60 (A), 8 (B)

OTHER SUBSTANCES

Simultaneous: acetaminophen (B), carbamazepine (B), phenobarbital (B), phenytoin (B), primidone (B), sulfapyridine (B)
KEYWORDS

serum
REFERENCE Ambrose,D.L.; Fntz,J.S. High-performance liquid chromatographic determination of drugs and metabolites in human serum and urine using direct injection and a unique molecular sieve, J.Chromatogr.B, 1998, 709, 89-96. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min.

Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 10.445
KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763, 149-163. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax KX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, na-

lorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994, 18, 233-242.

Barnidipine
Molecular formula: C27H29N3O6 Molecular weight: 491.54 CAS Registry No.: 104713-75-9 Merck Index: 1031

SAMPLE

Matrix: bile, urine Sample preparation: Inject urine and bile directly. Hydrolyse urine or bile by heating a 1 mL aliquot with 200 JJLL 200 mM pH 5.5 sodium acetate buffer and 20 |xL 100 U/mL Pglucuronidase at 37 for 24 h, add to a Sep-Pak C18 SPE cartridge, elute with MeOH. Evaporate the eluate, reconstitute the residue with MeOH:50 mM pH 5.0 sodium acetate buffer 5:95, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Nucleosil 5C18 Mobile phase: Gradient. MeOH:50 mM pH 5.0 sodium acetate buffer 5:95 for 5 min, to 50: 50 over 95 min Flow rate: 0.8 Detector: UV
OTHER SUBSTANCES Extracted: metabolites KEYWORDS

rat; dog; SPE

REFERENCE Teramura,T.; Tokunaga,T.; Matsumoto,H.; Watanabe,T.; Higuchi,S. Metabolism of barnidipine hydrochloride, a potent calcium antagonist, in rat and dog, Xenobiotica, 1996, 26, 177-187. SAMPLE

Matrix: blood Sample preparation: 500 |JLL Serum or plasma + 500 |xL 500 mM pH 11 phosphate buffer + 3 mL benzene (Caution! Benzene is a carcinogen!), shake mechanically for 5 min, centrifuge at 3000 rpm for 10 min. Remove 2.7 mL of the organic layer and add it to 50 ng IS, evaporate to dryness under a stream of nitrogen, reconstitute the residue in 75 JLJLL mobile phase, inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4 5 |xm Aluspher RP-select B (Merck) Mobile phase: MeCN:100 mM pH 11.8 Britton-Robinson buffer 40:60 Flow rate: 0.9 Injection volume: 5 Detector: E, JASCO Model 840-EC, glassy carbon working electrode 0.6 V, Ag/AgCl reference electrode
CHROMATOGRAM Retention time: 8.5

Internal standard: 2-(N-benzyl-N-methylamino) ethylmethyl l,4-dihydro-2,6-dimethyl-4phenyl-3,5-pyridinedicarboxylate hydrochloride (YC-204, Yamanouchi) (7.3) Limit of detection: 1 ng/mL

OTHER SUBSTANCES

Noninterfering: acetazolamide, allopurinol, amitriptyline, chlorpromazine, clonidine, cyproheptadine, hydrochlorothiazide, imipramine, methyldopa, phenoxybenzamine, propranolol, ascorbic acid
KEY WORDS

human; dog; serum; plasma; protect from light; pharmacokinetics

REFERENCE Takamura,K; Abdel-Wadood,H.M.; Kusu,R; RafaatJ.H.; Saleh,G.A.; El-Rabbat,N.A.; Otagiri,M. Determination of barnidipine in human serum and dog plasma by HPLC with electrochemical detection, Biol.Pharm.BulL, 1995, 18, 1311-1314. SAMPLE

Matrix: blood, enzyme incubations Sample preparation: 1 mL Enzyme incubation or plasma + 5 mL diethyl ether + 500 jxL saturated sodium bicarbonate, extract, centrifuge at 800 g for 5 min. Remove the organic layer and evaporate it to dryness, reconstitute the residue in 100 |xL MeOH, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Nucleosil 5C18 Mobile phase: MeOH:20 mM pH 3.0 bromo-tetra-n-propylammonium phosphate 50:50 Flow rate: 1 Detector: UV OTHER SUBSTANCES Extracted: metabolites
KEY WORDS

rat; dog; plasma

REFERENCE Teramura,T.; Tokunaga,T.; Matsumoto,H.; Watanabe,T.; Higuchi,S. Metabolism of barnidipine hydrochloride, a potent calcium antagonist, in rat and dog, Xenobiotica, 1996, 26, 177-187. SAMPLE

Matrix: microsomal incubations Sample preparation: 500 |xL Microsomal incubation + 500 |xL ice-cold MeOH, vortex, centrifuge at 1000 g for 15 min, filter (0.22 |xm), inject an aliquot of the filtrate.
HPLC VARIABLES

Column: 250 X 4.6 Nucleosil 5C18 Mobile phase: Gradient. MeCN:20 mM pH 7.0 ammonium acetate buffer 30:70 for 5 min, to 70:30 over 55 min, maintain at 70:30 for 30 min. Flow rate: 1 Detector: radioactivity
CHROMATOGRAM R e t e n t i o n time: 66 OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

rat; dog; liver; 14C labeled

REFERENCE Teramura,T.; Tokunaga,T.; Matsumoto,H.; Watanabe,T.; Higuchi,S. Metabolism of barnidipine hydrochloride, a potent calcium antagonist, in rat and dog, Xenobiotica, 1996, 26, 177-187.

Beclobrate
Molecular formula: C2OH23CIO3 Molecular weight: 346.85 CAS Registry No.: 55937-99-0 Merck Index: 1046

SAMPLE

Matrix: blood Sample preparation: 250 |JLL Plasma + 250 |xL acetic acid + 1.75 mL 10 |xg/mL IS in hexane, shake for 5 min, centrifuge at 2000 rpm for 20 min, inject a 50 JJLL aliquot of the supernatant.
HPLC VARIABLES

Guard column: 30 x 4 30-40 |xm RP18 (E. Merck) Column: 125 x 4 5 ^m LiChrosorb RP18 Mobile phase: MeCN:MeOH:water:acetic acid 40:30:30:0.1 Flow rate: 2 Injection volume: 50 Detector: UV 27
CHROMATOGRAM

Retention time: 4.1 (beclobric acid) Internal standard: SGD 2774 (5.8)
KEYWORDS

plasma; pharmacokinetics; bioequivalence

REFERENCE Gikalov,L; Ifflaender,U. Pharmacokinetik und Bioaquivalenz von zwei peroralen Beclobrat-Zubereitungen [Pharmacokinetics and bioequivalence of two peroral beclobrate preparations], Arzneimittelforschung, 1987, 37, 1065-1068. SAMPLE

Matrix: blood, urine Sample preparation: 200 |xL Plasma or 100 |xL urine + 500 (plasma) or 300 (urine) mg NaCl + 50 fiL 1 (xg/mL clofibric acid in MeOH + 1 (plasma) or 0.3 (urine) mL pH 4 buffer + 5 mL n-hexane:EtOH 90:10, shake horizontally for 10 min, centrifuge. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 55, add 50 IxL toluene and evaporate it to remove traces of water. Reconstitute the residue in 500 JULL dichloromethane, add 50 jxL 1 mg/mL 1-hydroxybenzotriazole in dichloromethane: pyridine 99:1, add 50 JJLL 1 mg/mL l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dichloromethane, add 50 |xL 1 mg/mL FLOPA, vortex, let stand at room temperature for 2 h, evaporate to dryness, reconstitute in 500 JJLL mobile phase, inject a 50 |xL aliquot. (FLOPA is the corresponding amine hydrochloride from (+)-(S)-flunoxaprofen. Synthesis is as follows (protect from light). 500 mg (+)-(S)-Flunoxaprofen in 50 mL dry toluene, slowly add 5 mL freshly distilled thionyl chloride, reflux for 1 h, evaporate to dryness under vacuum, dry the acyl chloride under vacuum over KOH for 2 days. Dissolve 0.5 mmoles acyl chloride in 5 mL acetone, add 600 mg sodium azide dissolved in ice water with stirring, stir at 0 for 30 min, add 10 mL ice-cold water, filter, dry solid in a desiccator under vacuum. Dissolve the solid in 1 mL toluene or dichloromethane (dried over 3 A molecular sieve), reflux for 10 min, evaporate, store resulting isocyanate under vacuum over a desiccant. Dissolve 0.5 mmole isocyanate in 5 mL acetone, add 20 mL 8.5% phosphoric acid, heat to 80 for 1.5 h, adjust to pH 13, extract with diethyl ether:dichloromethane 4:1. Wash the organic layer twice with water, dry over anhydrous sodium sulfate,

evaporate to dryness, dissolve in 1 mL toluene, evaporate to give crystals (mp 91). Dissolve in ether, add 0.5 M HCl in ether, filter, dissolve solid in a small volume of MeOH, precipitate with ether, dry FLOPA over phosphorus pentoxide under vacuum (Pharm.Res. 1990, 7, 1262).)
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Zorbax SiI Mobile phase: Gradient. A was n-hexane:chloroform:EtOH 100:10:0.75. B was n-hexane: chloroform:EtOH 100:10:20. A:B 100:0 for 10 min, 50:50 for 5 min, 100:0 for 5 min (stepwise). Flow rate: 2 Injection volume: 50 Detector: F ex 305 em 355 CHROMATOGRAM Retention time: 6 (-), 6.5 (+) Internal standard: clofibric acid (8) Limit of detection: 25 ng/mL
KEYWORDS

plasma; pharmacokinetics; chiral; derivatization; normal phase

REFERENCE Mayer,S.; Mutschler,E.; Spahn-Langguth,H. Pharmacokinetic studies with the lipid-regulating agent beclobrate: enantiospecific assay for beclobric acid using a new fluorescent chiral coupling component (S-FLOPA), Chirality, 1991, 3, 35-42.

Beclomethasone
Molecular formula: C22H29CIO5 Molecular weight: 408.92 CAS Registry No.: 4419-39-0, 5534-09-8 (beclomethasone dipropionate) Merck Index: 1047

SAMPLE

Matrix: blood Sample preparation: Add 30 mL EtOH to 50 mL plasma, extract with dichloromethane for 15 min, centrifuge at 2500 rpm for 5 min. Evaporate dichloromethane layer to dryness under vacuum at 30, reconstitute residue in mobile phase and inject a 100 fiL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jxm Alltima C18 (Alltech Associates, Australia) (A) or 250 X 4.6 5 jim Econosphere C18 (Alltech Associates, Australia) (B) or 150 X 4.6 5 |xm Ultrasphere C8 (C) Mobile phase: MeOH:MeCN:acetic acid:water 30.9:14.6:4.4:0.1 (A) or 25:20.5:4.4:0.1 (B) or 27.5:18:4.4:0.1 (C) Flow rate: 1.3 (A, C) or 1.2 (B) Injection volume: 100 Detector: UV 242
CHROMATOGRAM

Retention time: 4.9 (A), 5.5 (B), 3.3 (C) (beclomethasone); 23.0 (A), 25.9 (B), 17.6 (C) (beclomethasone dipropionate)
OTHER SUBSTANCES

Extracted: degradation products

KEYWORDS

plasma
REFERENCE Foe,K; Cheung,H.T.A.; Tattam,B.N.; Brown,K.R; Seale,J.P. Degradation products of beclomethasone dipropionate in human plasma, Drug Metab.Dispos., 1998, 26, 132-137. SAMPLE

Matrix: blood Sample preparation: 200 jxL Plasma + 300 (xL water + 6 mL diethyl ether, shake for 5 min, centrifuge at 1000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen. Dissolve in 100 |xL MeOH. Add 20 |xL copper acetate solution and let stand at room temperature for 1 h. Add 80 jxL diaminophthalhydrazide solution, heat at 80 for 110 min, cool, inject a 20 |xL aliquot. (Prepare copper acetate solution by dissolving 700 mg copper(II) acetate in 10 mL water, dilute to 100 mL with MeOH, discard after 1 month. Diaminophthalhydrazide solution was 7.5 mM 4,5-diaminophthalhydrazide in 3.5 M hydrochloric acid containing 625 mM (3-mercaptoethanol, discard after 5 h. Prepare 4,5-diaminophthalhydrazide dihydrochloride as follows. Reflux 316 g 4-nitrophthalic acid and 50 mL concentrated sulfuric acid in 500 mL MeOH for 10 h, recrystallize the product (dimethyl 4-nitrophthalate) from MeOH (mp 64-65E). Hydrogenate 47.8 g dimethyl 4-nitrophthalate in 300 mL MeOH over 13 g 5% platinum on carbon at an initial hydrogen pressure of 50 psi. When the calculated amount of hydrogen has been absorbed remove the catalyst and evaporate to dryness under reduced pressure, recrystallize the residue from aqueous MeOH to give dimethyl 4-aminophthalate (mp 83-84E). Stir 146.3 g dimethyl 4-aminophthalate in 1.4 L acetic anhydride at 60-70E for 2 h then leave over-

night, precipitate product with MeOH. Dry the product and rinse it with sodium carbonate solution, re-dry, recrystallize from benzene/MeOH (Caution! Benzene is a carcinogen!) to give dimethyl 4-acetamidophthalate (mp 138-140E). Add 100.4 g to 600 mL fuming (90%) nitric acid at 0-5E over 30 min, stir at 5-10E for 2.5 h, mix the reaction mixture with 800 mL cold dichloromethane, shake with crushed ice. Remove the organic layer and extract the aqueous layer with 200 mL cold dichloromethane. Combine the organic layers and wash them with ice water, cold sodium bicarbonate solution, and cold water. Dry over anhydrous magnesium sulfate, evapor ate to dryness under reduced pressure and, recrystallize repeatedly from MeOH to give dimethyl 4-acetamido-5-nitrophthalate (mp 123124.5E). Hydrolyze dimethyl 4-acetamido-5-nitrophthalate to dimethyl 4-amino-5-nitrophthalate. Hydrogenate 20.3 g dimethyl 4-amino-5-nitrophthalate in 250 mL MeOH over 1 g 5% platinum on carbon at an initial hydrogen pressure of 50 psi, remove the catalyst, evaporate to dryness under reduced pressure at 25E, recrystallize from chloroform/dichloromethane to give dimethyl 4,5-diaminophthalate (mp 111.5-113E). Add 1.1 g dimethyl 4,5-diaminophthalate to 3 mL hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) and 3 mL triethylamine in 20 mL MeOH, concentrate the resulting solution, triturate with benzene/MeOH, recrystallize from N,N'-dimethylacetamide/acetic acid to give 4,5-diaminophthalhydrazide (6,7-diamino-2,3-dihydrophthalazine-l,4-dione) (mp 407E) (J. Heterocycl. Chem. 1973, 10, 891), mix 4,5-diaminophthalhydrazide with a small amount of concentrated HCl, recrystallize from EtOH to give 4,5-diaminophthalhydrazide dihydrochloride.)
HPLC VARIABLES

Column: 250 X 4.6 5 ^m TSKgel ODS-120T (Tosoh, Japan) Mobile phase: MeCN:tetrahydrofuran:100 mM pH 7.0 phosphate buffer 24:3:73 Flow rate: 1.0 Injection volume: 20 Detector: Chemiluminiscence following post-column reaction. The column effluent mixed with 20 mM hydrogen peroxide in water pumped at 1.0 mL/min and then with 30 mM potassium hexacyanoferrate(III) in 3.0 M NaOH pumped at 2.0 mL/min and flowed to the detector.
CHROMATOGRAM

Retention time: 52 Internal standard: beclomethasone

OTHER SUBSTANCES

Extracted: dexamethasone Simultaneous: aldosterone, corticosterone, cortisone, 11-deoxycortisol, hydrocortisone, 18hydroxycorticosterone, 18-hydroxydeoxycorticosterone, prednisolone, prednisone Noninterfering: androstendione, cholesterol, estrone, estradiol, estriol, pregnenolone, progesterone Interfering: betamethasone.
KEYWORDS

plasma; derivatization; beclomethasone is IS

REFERENCE Ishida,J.; Sonezaki,S.; Yamaguchi,M.; Yoshitake,T., Determination of dexamethasone in plasma by highperformance liquid chromatography with chemiluminiscence detection, Anal.ScL, 1993, 9, 319-322. SAMPLE

Matrix: blood Sample preparation: Mix 1 mL plasma with 750 |xL 40 |mg/mL IS in EtOH, add 8 mL dichloromethane, extract on a roller mixer for 30 min. Centrifuge at 2500 rpm for 10 min at 25, collect the organic layer, evaporate to dryness at 30 under a stream of nitrogen. Reconstitute the residue in 1 mL mobile phase, centrifuge at 15000 rpm for 2 min, inject
a 100 J L J L L aliquot.

HPLC VARIABLES

Column: 250 X 4.6 Alltima C18 (Alltech) Mobile phase: MeCN:MeOH:water:glacial acetic acid 8.8:65:26.2:0.175 Flow rate: 1.3 Injection volume: 100 Detector: UV 242
CHROMATOGRAM

Retention time: 15.4 (dipropionate) Internal standard: dexamethasone-21-acetate (5.2) OTHERSUBSTANCES Extracted: degradation products
KEYWORDS

plasma; serum
REFERENCE Foe,K; Brown,K.E; Seale,J.P. Decomposition of beclomethasone propionate esters in human plasma, Biopharm.Drug Dispos., 1998, 19, 1-8. SAMPLE

Matrix: blood Sample preparation: 50 |xL Plasma + 100 |xL 20 |xg/mL cloprednol + 3 mL ether, shake 10 min, centrifuge at 3000 g, remove the organic phase and evaporate it to dryness under nitrogen. Take up the residue in 200 |xL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: Nucleosil R 10 C 18 Mobile phase: MeOH:MeCN:water:acetic acid 400:100:200:1 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Internal standard: cloprednol Limit of detection: 500 ng/mL

KEY WORDS

for beclomethasone dipropionate; plasma

REFERENCE Wurthwein,G.; Rohdewald,P. Activation of beclomethasone dipropionate by hydrolysis to beclomethasone-17-monopropionate, Biopharm.Drug Dispos., 1990, 11, 381-394. SAMPLE

Matrix: blood, tissue Sample preparation: Acidify plasma or lung tissue homogenate to pH 2 with 500 mM HCl, add 100 fiL 20 |xg/mL IS, extract with 8 mL dichloromethane. Evaporate the organic layer to dryness under vacuum, reconstitute in 120 |xL MeOH:5% acetic acid 50:50, inject an 80 |xL aliquot.
HPLC VARIABLES

Column: 250 x 4.6 5 \xm Zorbax ODS C18 Mobile phase: MeCN:MeOH:water 44:11:45 Flow rate: 1 Injection volume: 80 Detector: UV 242 or radioactivity

CHROMATOGRAM

Internal standard: hydrocortisone 21-S-propionate (JO 498)

OTHER SUBSTANCES

Extracted: metabolites, budesonide

KEYWORDS

for beclomethasone dipropionate; plasma; rat; lung; radiolabeled; pharmacokinetics

REFERENCE Chanoine,R; Grenot,C; Heidmann,R; Junien,J.L. Pharmacokinetics of butixocort 21-propionate, budesonide, and beclomethasone dipropionate in the rat after intratracheal, intravenous, and oral treatments, Drug Metab.Dispos., 1991, 19, 546-553. SAMPLE

Matrix: ileostomy effluent Sample preparation: Dilute ileostomy effluent 1:2 by weight with water and mix with 100 IxL 11 |xg/mL 17-hydroxyprogesterone. Extract 3 g aliquot three times with 10 mL dichloromethane by shaking for 1 min and centrifuging at 2000 rpm for 2 min. Wash combined extracts successively with 2 mL 0.1 M NaOH and 4 mL water by shaking for 30 s and centrifuging for 1 min then dry the organic layer under air at 40. Take up the extract in 1 mL MeOH, add 1.1 mL water and apply to C18 Bond Elut SPE cartridge. Wash with 10 mL water, wash with 5 mL MeOHrwater 45:55, elute with 2 mL MeOH. Add 50 (xL 20 |xg/mL progesterone to the eluate, dry at 40, take up in 100 jxL MeOH, inject 10 |xL aliquot.
HPLC VARIABLES

Guard column: Bondapak C18/Corasil Column: 300 X 3.9 ixBondapak C18 Mobile phase: MeOH:50 mM pH 3.0 sodium phosphate buffer 55:45 Flow rate: 3 Injection volume: 10 Detector: UV 254 and 238
CHROMATOGRAM

Retention time: 21.3 (beclomethasone dipropionate) Internal standard: 17-Hydroxyprogesterone (6.0) and progesterone (11.6)
OTHER SUBSTANCES

Extracted: beclomethasone alcohol, beclomethasone 17-monopropionate

KEYWORDS

SPE
REFERENCE Levine,D.S.; Raisys,V.A.; Ainardi,V. Coating of oral beclomethasone dipropionate capsules with cellulose acetate phthalate enhances delivery of topically active antiinflammatory drug to the terminal ileum, Gastroenterology, 1987, 92, 1037-1044. SAMPLE

Matrix: tissue Sample preparation: 100 mg Tissue + 2 mL Ringer's pH 6.8 phosphate buffer + 2 mL EtOH, centrifuge, wash residue twice. Pool supernatant and washings and evaporate to dryness, take up in 400 |xL EtOH, inject an aliquot.
HPLC VARIABLES

Guard column: Used but not specified

Column: ixBondapak C18 Mobile phase: Gradient. MeOH:water 40:60 to 80:20, time not specified

Flow rate: 1.5 Detector: UV 254

OTHER SUBSTANCES Extracted: beclomethasone monopropionate, beclomethasone, cyclomethasone KEYWORDS for beclomethasone dipropionate; lung REFERENCE Ronca-Testoni,S. Hydrolysis of cyclomethasone by the human lung, Int.J.Clin.Pharmacol.Res., 1983, 3, 17-20.

Befunolol
Molecular formula: C16H21NO4 Molecular weight: 291.35 CAS Registry No.: 39552-01-7 Merck Index: 1050

SAMPLE

Matrix: perfusate Sample preparation: 50 |xL Perfusate + 50 jxL pH 7.4 phosphate-buffered saline or 100 mM HCl + 100 |xL 50 jxg/mL salicyl methionine in MeOH, centrifuge at 12000 g for 10 min, inject a 50 jxL aliquot of the supernatant.
HPLC VARIABLES

Column: 150 X 4.6 Cosmosil 5C18-P (Nacalai Tesque) Mobile phase: MeOH:50 mM NaH2PO4 45:55 Flow rate: 1 Injection volume: 50 Detector: F ex 300 em 500
CHROMATOGRAM

Internal standard: salicyl methionine

KEY WORDS

rabbit
REFERENCE Sasaki,H.; Igarashi,Y.; Nagano,T.; Nishida,K; Nakamura,J. Different effects of absorption promoters on corneal and conjunctival penetration of ophthalmic (3-blockers, Pharm.Res., 1995, 12, 1146-1150. SAMPLE

Matrix: solutions Sample preparation: 50 JLJLL Solution + 50 |xL pH 7.4 PBS + 100 jxL 50 jjig/mL salicylmethionine in MeOH, centrifuge at 12000 g for 10 min, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 Cosmosil 5C18-P (Nacalai Tesque) Mobile phase: MeOH:50 mM NaH2PO4 45:55 Flow rate: 1 Injection volume: 50 Detector: F ex 300 em 500
CHROMATOGRAM

Internal standard: salicylmethionine

KEY WORDS

buffer; earle's balanced salt solution

REFERENCE Sasaki,H; Igarishi,Y; Nishida,K.; Nakamura,J. Intestinal permeability of ophthalmic (3-blockers for predicting ocular permeability, J.Pharm.ScL, 1994, 83, 1335-1338.

Benactyzine
Molecular formula: C20H25NO3 Molecular weight: 327.42 CAS Registry No.: 302-40-9, 57-37-4 (HCI) Merck Index: 1055 Lednicer No.: 1 93 SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mIVL 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 2.3 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,

protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. IL Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225. SAMPLE

Matrix: solutions Sample preparation: Prepare a 1 mg/mL solution in MeOH, inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Lichrosphere cyanopropyl Mobile phase: Carbon dioxide:MeOH:isopropylamine 90:10:0.05 Column temperature: 50 Flow rate: 3 Injection volume: 5 Detector: UV 220
CHROMATOGRAM Retention time: 1.99 OTHER SUBSTANCES

Simultaneous: buclizine, hydroxyzine, perphenazine, thioridazine, amitriptyline, desipramine, imipramine, nortriptyline, protriptyline
KEYWORDS

SFC; pressure 200 bar

REFERENCE Berger,T.A.; Wilson,W.H. Separation of drugs by packed column supercritical fluid chromatography. 2. Antidepressants, J.Pharm.ScL, 1994, 83, 287-290.

Benazepril
Molecular formula: C24H28N2O5 Molecular weight: 424.50 CAS Registry No.: 86541-75-5, 86541-74-4 (HCI) Merck Index: 1058 Led nicer No.: 5 135

SAMPLE

Matrix: formulations Sample preparation: Add MeOH:water 50:50 to powdered capsules or tablets so as to give a benazepril concentration of ca. 20 |xg/mL, stir for 15 min, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.5 5 |xm Hypersil ODS Mobile phase: MeCN:THF:20 mM pH 2.5 sodium heptanesulfonate 45.6:2.4:52 Flow rate: 1 Injection volume: 20 Detector: UV 215 CHROMATOGRAM Retention time: 15.5 OTHER SUBSTANCES Simultaneous: ramipril
KEYWORDS

capsules; tablets
REFERENCE Bonazzi,D.; Gotti,R.; Andrisano,V.; Cavrini,V. Analysis of ACE inhibitors in pharmaceutical dosage forms by derivative UV spectroscopy and liquid chromatography (HPLC), J.Pharm.Biomed.Anal., 1997,16, 431-438. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCNtwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JJIL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min)

Injection volume: 10-30 Detector: UV 205.2 CHROMATOGRAM Retention time: 17.003

KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: formulations Sample preparation: Grind tablets to a fine powder. Weigh out an amount equivalent to 25 mg benazepril, extract with MeOH, filter. Mix 100-500 |xL filtrate with 200 |JLL 4 mg/ mL IS in MeOH, make up to 10 mL with MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4 10 jxm LiChrosorb RP-18 Mobile phase: MeCN:buffer 30:70 (Buffer was 67 mM KH2PO4 adjusted to pH 2.4 with phosphoric acid.) Flow rate: 1 Injection volume: 20 Detector: UV 211 CHROMATOGRAM Retention time: 18.53 Internal standard: enalapril (8.58) Limit of detection: 5 jxg/mL Limit of quantitation: 10 jig/mL OTHER SUBSTANCES Simultaneous: cilazapril
KEYWORDS

tablets
REFERENCE GumieniczekjA.; Przyborowski,L. Determination of benazepril and cilazapril in Pharmaceuticals by high performance liquid chromatography, J.Liq.Chromatogr.ReLTechnoL, 1997, 20, 2135-2142.

Bendroflumethiazide
Molecular formula: C15H14F3N3O4S2 Molecular weight: 421.42 CAS Registry No.: 73-48-3 Merck Index: 1064 Lednicer No.: 2 358

SAMPLE

Matrix: blood Sample preparation: 3 mL Plasma + 2 mL 10 mM NaOH + 2 mL 10 mM HCl, mix, add 10 mL diethyl ether, shake gently on a platform shaker for 15 min, centrifuge at -10 at 2200 g for 15 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 70 for 45 min (these conditions are required to remove residual benzyl alcohol that is present as a preservative in the heparin), reconstitute the residue in 50 |xL 10 mM NaOH, vortex for 25 s, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 jrni jiBondapak C18 Mobile phase: Isopropanol:water:acetic acid 17:82:1 Flow rate: 2 Injection volume: 20 Detector: UV 269
CHROMATOGRAM

Retention time: 14.4 Internal standard: bendroflumethiazide OTHER SUBSTANCES Extracted: trichlormethiazide
KEYWORDS

plasma; silanize glassware; bendroflumethiazide is IS

REFERENCE Meyer,M.C; Hwang,P.T.R. Determination of trichlormethiazide in human plasma and urine by highperformance liquid chromatography, J.Chromatogr., 1981, 223, 466-472. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 fxm Symmetry C8 (Waters)

Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 208.7 CHROMATOGRAM Retention time: 18.632
KEYWORDS whole blood REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

SAMPLE Matrix: bulk Sample preparation: Dissolve in solvent, inject an aliquot. (Solvent was 750 mg KCl in 10 mL 1 M HCl, add 400 mL water, add 400 mL MeOH, make up to 1 L with water.)
HPLC VARIABLES Guard c o l u m n : 5 x 4 7 |jim Nucleosil-100 phenyl Column: 300 X 4 7 |xm Nucleosil-100 phenyl Mobile p h a s e : MeOH:water 40:60

Column temperature: 35 Flow rate: 1.5 Injection volume: 50 Detector: UV 270 CHROMATOGRAM Retention time: 10.5
OTHER SUBSTANCES

Simultaneous: hydroflumethiazide, degradation products

REFERENCE Frontini,R.; Mielck,J.B. Determination and quantitation of bendroflumethiazide and its degradation products using HPLC, J.Liq.Chromatogr., 1992, 15, 2519-2528. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Chirex 3001 (Phenomenex) Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 55:35:10 (EtOH/trifluoroacetic acid was premixed 20:1.) Flow rate: 1 Injection volume: 20 Detector: UV 272
CHROMATOGRAM

Retention time: 19, 21.5 (enantiomers)

KEY WORDS

chiral
REFERENCE Cleveland,!*. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical racemates, J.Liq.Chromatogr., 1995, 18, 649-671. SAMPLE

Matrix: solutions Sample preparation: Prepare a solution in MeOH:water 80:20, inject a 6 |xL aliquot.
HPLC VARIABLES

Guard column: 5 X 4 10 |xm LiChrosorb RP-8 Column: 100 X 4.6 5 jim Spheri RP-18 (Brownlee) Mobile phase: MeOH:water 80:20 containing 2 g/L lithium perchlorate Flow rate: 0.5 Injection volume: 6 Detector: E, ESA Model 5100A Coulochem, model 5020 guard cell +950 mV, Model 5010 analytical cell + 400 mV, palladium reference electrode, following post-column photolysis. The effluent from the column flowed through a 20 m X 0.3 mm coil of PTFE tubing and was irradiated at 254 nm with a Sylvania GTE 8 W low-pressure lamp.
CHROMATOGRAM

Retention time: 5 Limit of detection: 267 ng/mL

OTHER SUBSTANCES

Also analyzed: butizide, chlorthalidone, ethacrynic acid, furosemide, hydrochlorothiazide

KEYWORDS

post-column reaction
REFERENCE Macher,M.; Wintersteiger,R. Improved electrochemical detection of diuretics in high-performance liquid chromatographic analysis by postcolumn on-line photolysis, J.ChromatogrA, 1995, 709, 257-264. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 cellulose 3,5-dimethylphenylcarbamate/10-undecenoate bonded to allylsilica Mobile phase: Heptane:isopropanol:diethylamine 80:20:0.1 Flow rate: 1 Injection volume: 1000 Detector: UV 254 CHROMATOGRAM Retention time: k' 8.34
KEYWORDS

chiral; a 1.17
REFERENCE Oliveros,L.; Lopez,R; Minguillon,C; Franco,R Chiral chromatographic discrimination ability of a cellulose 3,5-dimethylphenylcarbamate/10-undecenoate mixed derivative fixed on several chromatographic matrices, J.Liq.Chromatogr., 1995, 18, 1521-1532.

SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 500 mg solid sodium bicarbonate, mix, add 2 mL 10 mM NaOH, mix, add 10 mL diethyl ether, shake gently on a platform shaker for 15 min, centrifuge at -10 at 2200 g for 15 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 70 for 45 min (these conditions are required to remove residual benzyl alcohol that is present as a preservative in the heparin), reconstitute the residue in 100 |xL MeOH, vortex for 25 s, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 |xm ixBondapak C18 Mobile phase: MeCNiMeOH:waterracetic acid 5:35:59:1 Flow rate: 2 Injection volume: 20 Detector: UV 280
CHROMATOGRAM

Retention time: 10.7 Internal standard: bendroflumethiazide

OTHER SUBSTANCES

Extracted: trichlormethiazide
KEYWORDS

silanize glassware; bendroflumethiazide is IS

REFERENCE Meyer,M.C; Hwang,P.T.R. Determination of trichlormethiazide in human plasma and urine by highperformance liquid chromatography, J.Chromatogr., 1981, 223, 466-472. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 2 mL 1 M pH 4.1 NaH2PO4 + 4 mL ethyl acetate, vortex for 2 min, centrifuge at 1500 g for 5 min. Remove the organic phase and add it to 5 mL 100 mM pH 7.5 Na2HPO4, vortex for 2 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 60, reconstitute the residue in 100 jxL MeCNrIO mM pH 3.0 phosphate buffer, inject a 5 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4 5 |xm LiCHrosorb RP-18 Mobile phase: Gradient. MeCNrIO mM pH 3.0 phosphate buffer 10:90 for 1.5 min then to 35:65 over 2 min Column temperature: 50 Flow rate: 1.5 Injection volume: 5 Detector: UV 271
CHROMATOGRAM

Retention time: 6.8 Limit of quantitation: 500 ng/mL

OTHER SUBSTANCES

Extracted: chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, clopamide, methyclothiazide, furosemide, metolazone, mefruside, cyclopenthiazide, bumetanide Simultaneous: indapamide, clorexolone, ethacrynic acid Noninterfering: aspirin, albuterol, allopurinol, alprenolol, atenolol, captopril, carbimazole, clonidine, coloxyl, danthron, diazepam, digoxin, doxepin, glibenclamide, hydralazine, in-

domethacin, labetalol, metformin, methyldopa, metoprolol, mianserin, minoxidil, nifedipine, nitrazepam, oxazepam, oxprenolol, pindolol, prazosin, propranolol, senokot, theophylline, trifluoperazine
REFERENCE Fullinfaw,R.O.; Bury,R.W.; Moulds,R.F.W. Liquid chromatographic screening of diuretics in urine, J.Chromatogr., 1987, 415, 347-356. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 0.5 g solid buffer I (pH 5-5.5), vortex 15 s, add 4 mL ethyl acetate, agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and vortex it with 2 mL 5% aqueous lead acetate for 10 s, centrifuge at 600 g for 5 min, remove and keep organic phase. 2 mL Urine + 0.5 g solid buffer II (pH 9-9.5), vortex 15 s, add 4 mL ethyl acetate, agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and combine it with previous organic layer. Evaporate to dryness at 50 under a stream of nitrogen, reconstitute in 300 (xL 50 |xg/mL p-hydroxyethyltheophylline in MeOH, inject 5 JULL aliquot. (Solid buffer I was KH2PO4:Na2HPO4 99:1, solid buffer II was NaHCO3:K2CO3 3:2.)
HPLC VARIABLES

Column: 250 X 4.6 5 |xm HP Hypersil ODS (A) or HP LiChrosorb RP-18 (B) Mobile phase: Gradient. MeCN:buffer from 15:85 at 2 min to 80:20 at 20 min (Buffer was 50 mM NaH2PO4 containing 16 mM propylamine hydrochloride, adjusted to pH 3 with concentrated phosphoric acid.) Flow rate: 1 Injection volume: 5 Detector: UV 230, UV 275
CHROMATOGRAM

Retention time: 15 (A), 15.4 (B) Internal standard: p-hydroxyethyltheophylline (3.7 (A), 4.4 (B)) Limit of detection: 1000 ng/mL
OTHER SUBSTANCES

Extracted: furosemide, metolazone, amiloride, acetazolamide, chlorothiazide, hydrochlorothiazide, quinethazone, triamterene, hydroflumethiazide, chlorthalidone, dichlorphenamide, trichloromethiazide, methyclothiazide, benzthiazide, cyclothiazide, ethacrynic acid, bumetanide, probenecid, spironolactone, canrenone, flumethiazide Noninterfering: acetaminophen, aspirin, caffeine, diflunisal, fenoprofen, ibuprofen, indomethacin, methocarbamol, naproxen, phenylbutazone, sulindac, tetracycline, theobromine, theophylline, tolmetin, trimethoprim, verapamil Interfering: polythiazide
REFERENCE Cooper,S.R; Masse,R.; Dugal,R. Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography, J.Chromatogr., 1989, 489, 65-88. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 1 mL 10 mM HCl + 2000 ng bendroflumethiazide, extract with 5 mL ethyl acetate, centrifuge at 3000 rpm for 5 min. Remove the organic layer and dry it under a stream of nitrogen at 40. Reconstitute with 100 |xL MeOH, inject a 2 |xL aliquot.
HPLC VARIABLES

Column: 100 X 2.1 5 um Hypersil ODS

Mobile phase: Gradient. MeOH: 50 mM ammonium acetate from 10:90 to 60:40 over 10 min, maintain at 60:40 for 10 min. Column temperature: 40 Flow rate: 0.3 Injection volume: 2 Detector: F ex 223 em 415 or UV 230
CHROMATOGRAM

Retention time: 8.6 Internal standard: bendroflumethiazide

OTHER SUBSTANCES

Extracted: bumetanide (F ex 231 em 426 or UV), furosemide (UV), piretanide (UV), cyclopenthiazide (UV), etozolin (UV), canrenone (UV)
KEYWORDS

bendroflumethiazide is IS
REFERENCE Gradeen,C.Y.; Billay,D.M.; Chan,S.C. Analysis of bumetanide in human urine by high-performance liquid chromatography with fluorescence detection and gas chromatography/mass spectrometry, JAnal.ToxicoL, 1990, 14, 123-126. SAMPLE

Matrix: urine Sample preparation: Make 5 mL urine alkaline (pH 9-10), add 2 g NaCl, extract twice with 6 mL ethyl acetate. Combine the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 200 jxL MeCN/water, inject a 10-20 jxL aliquot.
HPLC VARIABLES

Column: 100 X 4 5 |xm SGE 100 GL-4 C18P (Scientific Glass Engineering) Mobile phase: MeCN:MeOH:water:trifluoroacetic acid 4.5:10.5:85:0.5 Flow rate: 0.8 or 1 Injection volume: 10-20 Detector: MS, ZAB2-SEQ (VG), PSP source coupled to LC, source 250, probe 240-260, scan m/z 200-550 or UV 270
CHROMATOGRAM Retention time: 9.2 OTHER SUBSTANCES

Extracted: amiloride, chlorthalidone, triamterene, furosemide, benzthiazide

REFERENCE Ventura,R.; Fraisse,D.; Becchi,M.; Paisse,O.; Segura,J. Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography-mass spectrometry in doping control, J.Chromatogr., 1991, 562, 723-736. SAMPLE

Matrix: urine Sample preparation: Direct injection.

HPLC VARIABLES

Guard column: 35 X 4 5 u.m Spherisorb ODS-2 Column: 120 X 4 5 |xm Spherisorb ODS-2 Mobile phase: MeOH:50 mM sodium dodecyl sulfate 5:95

Column temperature: 50 Flow rate: 1 Injection volume: 20 Detector: UV 224

CHROMATOGRAM

Retention time: 15.2 Limit of detection: 500 ng/mL OTHER SUBSTANCES Simultaneous: chlorthalidone
REFERENCE Bonet Domingo,E.; Medina Hernandez,M.J.; Ramis Ramos,G.; Garcia Alvarez-Coque,M.C. High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography, J.Chromatogr., 1992, 582, 189-194. SAMPLE

Matrix: urine Sample preparation: Buffer urine to 4.9 by mixing with an equal volume of pH 4.9 200 mM sodium phosphate buffer. Inject a 40 JJLL aliquot onto column A with mobile phase A, after 3 min backflush the contents of column A onto column B with mobile phase B and start the gradient. At the end of the run re-equilibrate for 10 min.
HPLC VARIABLES

Column: A 20 X 4 5 |xm Hypersil octadecylsilica ODS; B 200 X 4.6 5 ^m Shiseido SG-120 polymer-based C18 Mobile phase: A water; B Gradient. MeCN:buffer from 7:93 to 15:85 over 3.5 min, to 50: 50 over 8.5 min, maintain at 50:50 for 11 min (Buffer was 6.9 g NaH2PO4-H2O in 1 L water, pH adjusted to 3.1 with phosphoric acid.) Flow rate: 1 Injection volume: 40 Detector: UV 270
CHROMATOGRAM

Retention time: 18.8 Limit of detection: 500 ng/mL

OTHER SUBSTANCES

Extracted: acetazolamide, amiloride, benzthiazide, bumetanide, caffeine, carbamazepine, chlorothiazide, chlorthalidone, clopamide, dichlorfenamide, ethacrynic acid, furosemide, hydrochlorothiazide, metyrapone, probenecid, spironolactone, triamterene, trichlormethiazide
KEYWORDS

column-switching; optimum detection wavelengths vary for each drug

REFERENCE Saarinen,M.; Siren,H.; Riekkola,M.-L. A column switching technique for the screening of diuretics in urine by high performance liquid chromatography, J.Liq.Chromatogr., 1993,16, 4063-4078. SAMPLE

Matrix: urine Sample preparation: 5 mL Urine + 50 jxL 100 |xg/mL 7-propyltheophylline in MeOH + 200 J U L L ammonium chloride buffer + 2 g NaCl, extract with 6 mL ethyl acetate by rocking at 40 movements/min for 20 min and centrifuging at 800 g for 5 min, repeat extraction, combine organic layers, evaporate to dryness at 40 under a stream of nitrogen. Recon-

stitute in 200 |xL MeCN:water 15:85 and inject 20 |xL aliquots. (Ammonium chloride buffer was 28 g ammonium chloride in 100 mL water with the pH adjusted to 9.5 with concentrated ammonia solution.)
HPLC VARIABLES

Column: 75 X 4.6 3 |xm Ultrasphere ODS Mobile phase: Gradient. MeCN: 100 mM ammonium acetate adjusted to pH 3 with concentrated phosphoric acid. From 10:90 to 15:85 over 2 min to 55:45 over 3 min to 60:40 over 3 min. Kept at 60:40 for 1 min, decreased to 10:90 over 1 min and equilibrated at 10:90 for 2 min. Flow rate: 1 Injection volume: 20 Detector: UV 270
CHROMATOGRAM

Retention time: 7.0 Internal standard: 7-propyltheophylline (4.5) Limit of detection: 50 ng/mL
OTHER SUBSTANCES

Simultaneous: xipamide, bumetanide, acetazolamide, amiloride, benzthiazide, buthiazide, caffeine, canrenone, chlorthalidone, clopamide, cyclothiazide, diclofenamide, furosemide, hydrochlorothiazide, mesocarb, morazone, piretanide, probenecid, spironolactone, torsemide, triamterene Interfering: polythiazide, ethacrynic acid
REFERENCE Ventura,R.; Nadal,T.; Alcalde,R; Pascual,J.A.; Segura,J. Fast screening method for diuretics, probenecid and other compounds of doping interest, J.ChromatogrA, 1993, 655, 233-242.

SAMPLE Matrix: urine Sample preparation: Direct injection into column A with mobile phase A for 1 min then back flush onto column B with mobile phase B.
HPLC VARIABLES

Column: A 20 X 2.1 30 p,m Hypersil ODS-C18; B 250 X 4 5 fjim Hypersil ODS-C18 Mobile phase: A Water; B Gradient. MeCNrbuffer 15:85 for 1.5 min then to 80:20 over 8 min. Keep at 80:20 for 2.5 min then re-equilibrate with 15:85. (Buffer was 50 mM NaH2PO4 +1.4 mL propylamine hydrochloride per liter adjusted to pH 3 with concentrated phosphoric acid.) Flow rate: 1 Injection volume: 50 Detector: UV 230
CHROMATOGRAM

Retention time: 9.8 Limit of detection: 20 ng/mL.

OTHERSUBSTANCES

Simultaneous: bumetanide, ethacrynic acid, acetazolamide, amiloride, chlorthalidone, cyclothiazide, furosemide, hydrochlorothiazide, probenecid, spironolactone, triamterene
REFERENCE Campins-Falco,R; Herraez-Hernandez,R.; Sevillano-Cabeza,A. Column-switching techniques for screening of diuretics and probenecid in urine samples, Anal.Chem., 1994, 66, 244-248.

Benfluorex
Molecular formula: C19H20F3NO2 Molecular weight: 351.37 CAS Registry No.: 23602-78-0, 23642-66-2 (HCI) Merck Index: 1066

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) JJLL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 fxm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 16.378 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Benidipine
Molecular formula: C28H31N3O6 Molecular weight: 505.57 CAS Registry No.: 105979-17-7 Merck Index: 1071

SAMPLE

Matrix: bile, blood, urine Sample preparation: Urine, bile. Inject urine and bile directly. Plasma. Add an equal volume of MeCN, centrifuge, remove supernatant and evaporate it to dryness. Reconstitute residue in MeCN: 10 mM pH 5 ammonium acetate 20:80.
HPLC VARIABLES

Column: 300 X 8 Unisil Q5C18 (Gaschro Kogyo) Mobile phase: Gradient. A was MeCN: 10 mM pH 5 ammonium acetate 20:80. B was MeCN: 10 mM pH 5 ammonium acetate 80:20. A:B from 100:0 to 92:8 over 4 min, to 83:17 over 4 min, to 75:25 over 4 min, to 63:37 over 4 min, to 55:45 over 8 min, to 50:50 over 8 min, to 42:58 over 4 min, to 0:100 over 9 min, hold at 0:100 for 25 min. Flow rate: 2 for 36 min, then 3 Detector: Radioactivity
OTHER SUBSTANCES

Extracted: metabolites
KEYWORDS

plasma; semi-preparative; rat; dog; radiolabelled

REFERENCE Kobayashi,H-; Okumura,S.; Kosaka,Y; Kobayashi,S.; Inoue,A-; Oka,T.; Nakamizo,N. Identification of benidipine hydrochloride metabolites in rats and dogs, Arzneimittelforschung, 1988, 38, 1753-1756. SAMPLE

Matrix: bulk Sample preparation: Dissolve 50 mg in 50 mL MeOH:water 70:30, remove a 10 mL aliquot and add it to 10 mL 1 mg/mL diphenylamine in MeOH, make this mixture up to 100 mL with MeOH:water 70:30, inject a 10 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 ixBondapak C18 Mobile phase: MeOH: 100 mM pH 5 ammonium acetate 70:30 (MeOH:50 mM pH 3.5 phosphate buffer:diisopropylamine 60:40:1 at 0.7 mL/min to detect other diastereomer) Flow rate: 1 Injection volume: 10 Detector: UV 236
CHROMATOGRAM

Retention time: 10 Internal standard: diphenylamine (6) OTHER SUBSTANCES Simultaneous: impurities

REFERENCE Suzuki,H.; Ono,E.; Ueno,H.; Takemoto,Y; Nakamizo,N. Physico-chemical properties and stabilities of the highly potent calcium antagonist benidipine hydrochloride, Arzneimittelforschung, 1988, 38, 1671-1676. SAMPLE

Matrix: formulations Sample preparation: Weigh out solid dispersion or solid dispersion granules equivalent to 20 mg benidipine hydrochloride, add 200 mL water, 100 mM hydrochloric solution, or Mcllvaine buffer (pH 4.0, 6.0), filter (0.2 |xm), dilute with MeOH, inject an aliquot.
HPLC VARIABLES

Column: C18 (YMC-Pack ODS-A) Mobile phase: MeOH:THF:50 mM pH 3.0 phosphate buffer 27:8:65 Flow rate: 1 Detector: UV 237 CHROMATOGRAM Internal standard: benzoin
KEYWORDS

solid dispersions; solid dispersion granules

REFERENCE Suzuki,H.; Miyamoto,N.; Masada,T.; Hayakawa,E.; Ito,K. Solid dispersions of benidipine hydrochloride. I. Preparations using different solvent systems and dissolution properties, Chem.Pharm.Bull., 1996, 44, 364-371. SAMPLE

Matrix: solutions Sample preparation: Direct injection of a MeOH solution containing 200-1000 ng.
HPLC VARIABLES

Column: 250 X 4.6 Sumchiral OA-4500 (Sumika Chemical Analysis Service) Mobile phase: n-Hexane:l,2-dichloroethane:MeOH:trifluoroaceticacid 250:140:10:1 Flow rate: 1 Detector: UV 254
CHROMATOGRAM

Retention time: 33 (+), 42(-) (a = 1.30)

KEYWORDS chiral REFERENCE Ohkubo,T.; Uno,T.; Sugawara,K. Enantiomer separation of dihydropyridine derivative calcium antagonists by high-performance liquid chromatography with chiral stationary phases, J.ChromatogrA, 1994, 659, 467-471.

Benoxaprofen
Molecular formula: C16H12CINO3 Molecular weight: 301.73 CAS Registry No.: 67434-14-4 Merck Index: 1075

SAMPLE

Matrix: bile, blood Sample preparation: 10 (xL Plasma, or bile + 180 |xL MeCN + 30 |xL 100 (xL/mL IS in DMSO + 30 |xL water, vigorously mix. Centrifuge at 15000 g for 10 min at 4, inject a 10 (ULL aliquot of the supernatant.
HPLC VARIABLES

Guard column: 10 X 4.6 SUMICHIRAL OA 2500 (Sumika Chemical Analysis Service, Japan) Column: 250 X 4.6 SUMICHIRAL OA 2500 (Sumika Chemical Analysis Service, Japan) Mobile phase: MeOH containing 40 mM ammonium acetate (plasma) or MeCN.MeOH: water 15:85:5 containing 10 mM ammonium acetate (bile) Flow rate: 1.0 Injection volume: 10 Detector: F ex 315, em 365
CHROMATOGRAM

Retention time: 10.3 (R, plasma) 11.8 (S, plasma), 22.5 (R, bile) 26.0 (S, bile) Internal standard: naproxen methyl ester (4.4) (plasma), (4.3) (bile) Limit of detection: 10 pg/mL OTHER SUBSTANCES Extracted: metabolites
KEY WORDS

plasma; rat; chiral; pharmacokinetics

REFERENCE Mohri,K.; Okada,K.; Benet,L.Z. Stereoselective metabolism of benoxaprofen in rats. Biliary excretion of benoxaprofen taurine conjugate and glucuronide, Drug Metab.Dispos., 1998, 26, 332-337. SAMPLE

Matrix: bile, blood, urine Sample preparation: 10 |xL Plasma, bile, or urine + 180 \xL MeCN + 30 |xL 100 |xL/mL IS in DMSO + 30 |xL water, mix vigorously. Centrifuge at 15000 g for 10 min at 4, inject a 10 |JLL aliquot of the supernatant.
HPLC VARIABLES

Guard column: 10 X 4.6 5 fjim Capcell Pak C18 (Shiseido, Japan) Column: 250 X 4.6 5 |xm Capcell Pak C18 (Shiseido, Japan) Mobile phase: MeCN:THF:10 mM tetrabutylammonium hydrogen sulfate buffer 35:35:100 Flow rate: 1.3 Injection volume: 10 Detector: F ex 315 em 365
CHROMATOGRAM

Retention time: 17.5 Internal standard: naproxen methyl ester (13.2)

Next Page
Limit of detection: 10 pg/mL OTHER SUBSTANCES Extracted: metabolites KEYWORDS plasma; rat; pharmacokinetics REFERENCE Mohri,K; Okada,K.; Benet,L.Z. Stereoselective metabolism of benoxaprofen in rats. Biliary excretion of benoxaprofen taurine conjugate and glucuronide, Drug Metab.Dispos., 1998, 26, 332-337.

Previous Page

Benperidol
Molecular formula: C22H24FN3O2 Molecular weight: 381.45 CAS Registry No.: 2062-84-2 Merck Index: 1077 LednicerNo.: 2 290

SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanolrn-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 |xm NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 246
CHROMATOGRAM

Retention time: 4.62 Limit of detection: <120 ng/mL

KEY WORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol;

aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromor amide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262. SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V CHROMATOGRAM Retention time: 1.8
OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, peri-

cyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Benzalkonium chloride
CAS Registry No.: 8001-54-5 Merck Index: 1086 SAMPLE

Matrix: formulations Sample preparation: Dilute with water, inject an aliquot.

HPLC VARIABLES

Column: 300 X 3.9 10 ^m jxBondapak CN Mobile phase: MeCN:buffer 50:50 (Buffer was 13.6 g/L sodium acetate, pH adjusted to, 4.5 with glacial acetic acid.) Flow rate: 1.5 Detector: UV 254
CHROMATOGRAM

Retention time: 8 (C12), 11 (C14), 14 (C16) OTHER SUBSTANCES Simultaneous: demecarium

KEYWORDS

ophthalmic solution
REFERENCE Cohn,L.J.; Greely,V.J.; Tibbetts,D.L. Determination of demecarium bromide and related compounds by high-performance liquid chromatography, J.Chromatogr., 1985, 321, 401-405. SAMPLE

Matrix: formulations
HPLC VARIABLES

Column: 300 X 3.9 10 urn LiChrosorb Si-60 Mobile phase: MeOH:water 60:40 containing 4 mM disodium citrate and 4 mM tetrabutylammonium bromide, pH 6.0 Flow rate: 1 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 8
OTHER SUBSTANCES

Simultaneous: domiphen bromide, thimerosal, xylometazoline

KEY WORDS

nasal drops
REFERENCE Lingeman,H.; van Munster,H.A.; Beynen,J.H.; Underberg,W.J.; Hulshoff,A. High-performance liquid chromatographic analysis of basic compounds on non-modified silica gel and aluminium oxide with aqueous solvent mixtures, J.Chromatogr., 1986, 352, 261-274.

SAMPLE

Matrix: formulations Sample preparation: Condition a C18 Sep-Pak SPE cartridge with THF:mobile phase 70: 30 then with water. Add 10 mL formulation to the SPE cartridge, wash with water, elute with 3 mL THF:mobile phase 70:30 then with 3 mL mobile phase, mix the eluates, inject a 100 |xL aliquot.
HPLC VARIABLES Column: 150 X 4.6 5 jxm Zorbax Stablebond CN Mobile p h a s e : THF:water:triethylamine 150:250:2, pH adjusted to 3.0 0 . 1 with phosphoric acid

Column temperature: 40 Flow rate: 2 Injection volume: 100 Detector: UV 215

CHROMATOGRAM

Retention time: 3.5 (C12), 6.5 (C14), 10 (C16)

KEYWORDS

SPE; eye
REFERENCE Elrod,L.,Jr.; Golich,T.G.; Morley,J.A. Determination of benzalkonium chloride in eye care products by high-performance liquid chromatography and solid-phase extraction or on-line column switching, J.Chromatogr., 1992, 625, 362-367. SAMPLE

Matrix: formulations Sample preparation: Condition a 1 mL Supelcoclean cyano SPE cartridge with 2 mL MeCN and 2 mL water. Add 4 mL formulation to the SPE cartridge, wash with 2 mL MeCNtbuffer 30:70, elute with 5 mL mobile phase, make up eluate to 10 mL with water, inject a 100 |xL aliquot. (Buffer was 6 mL concentrated phosphoric acid in 1950 mL water, adjust pH to 5.0 with 50% NaOH, make up to 2 L with water.).
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Ultrasphere cyano Mobile phase: MeCN:buffer 60:40 (Buffer was 6 mL concentrated phosphoric acid in 1950 mL water, adjust pH to 5.0 with 50% NaOH, make up to 2 L with water.) Flow rate: 2 Injection volume: 100 Detector: UV 210
CHROMATOGRAM

Retention time: 4.5 (ClO), 5.5 (C12), 6.5 (C14), 7.5 (C16), 8.5 (C18) OTHER SUBSTANCES Also analyzed: tyloxapol
KEY WORDS

ophthalmic solutions; eye; SPE

REFERENCE Fan,T.Y.; Wall,G.M. Determination of benzalkonium chloride in opthalmic solutions containing tyloxapol by solid-phase extraction and reversed-phase high-performance liquid chromatography, J.Pharm.ScL, 1993, 82, 1172-1174.

SAMPLE

Matrix: formulations Sample preparation: 2 mL Sample + 1 mL 200 (xg/mL emetine hydrochloride in water, make up to 10 mL with mobile phase, filter (0.45 |xm), inject a 50-100 (JLL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 5 jjiin Technosphere RP C-8 (HPLC Technology) Mobile phase: MeCN:40 mM tetramethylammonium bromide: 1 M acetic acid 80:15:5 (apparent pH 4.5) Flow rate: 1.5 Injection volume: 50-100 Detector: UV 260
CHROMATOGRAM

Retention time: 3.15 (C12), 4.21 (C14), 5.78 (C16) Internal standard: emetine (1.75) Limit of quantitation: 10 |xg/mL
OTHER SUBSTANCES

Simultaneous: naphazoline, tetrahydrozoline

KEYWORDS

nasal; ophthalmic
REFERENCE Santoni,G.; Medica,A.; Gratteri,R; Furlanetto,S.; Pinzauti,S. High-performance liquid chromatographic determination of benzalkonium and naphazoline or tetrahydrozoline in nasal and ophthalmic solutions, Farmaco, 1994, 49, 751-754. SAMPLE

Matrix: formulations Sample preparation: Inject a 50 (JLL aliquot.

HPLC VARIABLES

Column: 300 X 3.9 10 fxm u-Bondapak phenyl Mobile phase: MeCN:buffer 65:35 (Buffer was 50 mM KH2PO4 and 57 mM sodium hexanesulfonate, adjusted to pH 6.3 with 1 M NaOH.) Flow rate: 1.8 Injection volume: 50 Detector: UV 215
CHROMATOGRAM

Retention time: 7.5 (C 12), 12.1 (C 14) OTHER SUBSTANCES Noninterfering: phenylephrine
KEYWORDS

ophthalmic solution; stability-indicating

REFERENCE Parhizkari,G.; Miller,R-B.; Chen,C. A stability-indicating HPLC method for the determination of benzalkonium chloride in phenylephrine HCl 10% ophthalmic solution, J.Liq.Chromatogr., 1995, 18, 553563. SAMPLE

Matrix: formulations

Sample preparation: Dilute 0.5% ophthalmic solution 1:5 with mobile phase, inject a 100 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 3 |xm CPS Hypersil-1 cyano Mobile phase: MeCN:buffer 65:35 (Buffer was 50 mM sodium propionate adjusted to pH 5.3 with concentrated sulfuric acid.) Flow rate: 1.3 Injection volume: 100 Detector: UV 214
CHROMATOGRAM

Retention time: 10.0 (C12), 11.7 (C14)

KEYWORDS

ophthalmic solutions; stability-indicating

REFERENCE Parhizkari,G.; Delker,G.; Miller,R.B.; Chen,C. A stability-indicating HPLC method for the determination of benzalkonium chloride in 0.5% Tramadol ophthalmic solution, Chromatographia, 1995, 40, 155158. SAMPLE

Matrix: sewage Sample preparation: Condition a C18ec SPE cartridge (Macherey-Nagel) with three bed volumes of MeOH and three bed volumes of water. Allow sewage to settle, add an 8 mL aliquot of the supernatant to the SPE cartridge, wash with 3 bed volumes of water, wash with 2 bed volumes of ethyl acetate, elute with 2 bed volumes of 1% calcium chloride in MeOH:ethyl acetate 50:50, inject a 15 U | LL aliquot of the eluate. (For all steps involving the SPE cartridge the flow rate should be 3 mL/min.)
HPLC VARIABLES

Column: 250 X 4 5 |xm Partisil PAC CN-NH2 Mobile phase: Chloroform:MeOH 80:20 Column temperature: 15 Flow rate: 1 Injection volume: 15 Detector: F ex 383 em 459 following post-column extraction. The column effluent mixed with 37 |xg/mL sodium 9,10-dimethoxyanthracene-2-sulfonate in water pumped at 0.3 mL/ min and the mixture flowed to a phase separator. The organic phase flowed to the detector and the aqueous phase was discarded. (9,10-Dimethoxyanthracene-2-sulfonate forms a hydrophobic fluorescent ion-pair with benzalkonium chloride.)
CHROMATOGRAM

Retention time: 2.49 Limit of detection: 50 ng/mL

OTHER SUBSTANCES

Simultaneous: dimethyldidecylammonium chloride (DDMAC)

KEYWORDS

post-column reaction; post-column extraction; SPE

REFERENCE Kummerer,K; Eitel,A; Braun,lL; Hubner,R; Daschner,E; Mascart,G.; Milandri,M.; Reinthaler,E; Verhoef,J. Analysis of benzalkonium chloride in the effluent from European hospitals by solid-phase extraction and high-performance liquid chromatography with post-column ion-pairing and fluorescence detection, J.ChromatogrA, 1997, 774, 281-286.

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm cyano Mobile phase: MeCN: 100 mM pH 5 sodium acetate buffer 60:40 Flow rate: 1 Detector: UV 254
KEYWORDS

comparison with capillary electrophoresis

REFERENCE Prince,S.J.; Allen,L.V. Analysis of benzalkonium chloride and its homologs: HPLC vs HPCE (Abstract APQ 1093), Pharm.Res., 1996, 13, S26-S26. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm cyano Mobile phase: MeCN: 100 mM pH 5 sodium acetate buffer 60:40 Flow rate: 1 Detector: UV 254
CHROMATOGRAM

Limit of quantitation: 25 jxg/mL

KEYWORDS

run time 25 min; for C12; C14; C16 homologs; comparison with capillary electrophoresis
REFERENCE Prince,S.J.; McLaury,H.J.; Allen,L.V.,Jr. Comparison of HPCE and HPLC for the separation and quantitation of benzalkonium chloride homologs (Abstract 3012), Pharm.Res., 1997, 14, S463-S464.

Benzbromarone
Molecular formula: C17H12Br2O3 Molecular weight: 424.09 CAS Registry No.: 3562-84-3 Merck Index: 1093 Lednicer No.: 2 354

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 /ULL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 204
CHROMATOGRAM Retention time: 26.075 KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Benzethidine
Molecular formula: C23H29NO3 Molecular weight: 367.49 CAS Registry No.: 3691-78-9

SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mIVL 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 2.1 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzocaine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ran-

itidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Benzethonium chloride
Molecular formula: C27H42CINO2 Molecular weight: 448.09 CAS Registry No.: 121-54-0 Merck Index: 1103

SAMPLE

Matrix: saliva Sample preparation: Collect sample on Periopaper strip (filter paper), add paper to 100 IxL MeCN:water:glacial acetic acid 55:44.8:0.2 containing 7 mM sodium lauryl sulfate, vortex for 1 min, sonicate for 20 min, vortex, inject a 50 |iL aliquot.
HPLC VARIABLES

Column: 100 X 2.1 5 |xm C18 ODS-B Exsil (HiChrome) Mobile phase: MeCN:0.2% acetic acid 55:45 containing 5 mM sodium lauryl sulfate Flow rate: 0.5 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 7 Internal standard: benzethonium OTHER SUBSTANCES Extracted: chlorhexidine
KEYWORDS

narrow-bore; benzethonium is IS
REFERENCE Medlicott,N.J.; Ferry,D.G.; Tucker,LG.; Rathbone,M.J.; Holborow,D.W.; Jones,D.S. High performance liquid chromatographic (HPLC) assay for the determination of chlorhexidine in saliva film, J.Liq.Chromatogr., 1994, 17, 1605-1620. SAMPLE

Matrix: tissue Sample preparation: Mix 10 g minced fish or squid + 50 mL 96% EtOH + 50 |xL 1-hexanesulfonic acid + 500 JULL 2 M HCl, mix thoroughly for 1 h, filter, extract residue with 50 mL 96% EtOH,filter, press filter cake, wash with 25 mL EtOH. Combine filtrates, evaporate under vacuum below 70, take up residue in 500 JJLL 2 M HCl + 500 |L MeOH + 50 \JU\J 1-hexanesulfonic acid + 5 mL light petroleum (bp 30-40), agitate for 2 min, pour out of flask, rinse flask with the same mixture, rinse flask with light petroleum. Combine all extracts, heat at 70 in a water bath until the organic layer disappears, add 2 mL dichloromethanerlight petroleum 50:50, mix for 1 min, inject a 5-20 JULL aliquot of the organic phase.
HPLC VARIABLES

Column: 300 X 3.9 fxBondapak CN Mobile phase: MeCN: 100 mM ammonium acetate 75:25, containing 15 mIVL PIC B6 (Waters) Flow rate: 1 Injection volume: 5-20 Detector: UV 254

CHROMATOGRAM

Retention time: 5.5 Limit of detection: 5-10 ppm

KEYWORDS

fish; squid
REFERENCE Reuvers,T.B.A.; Ortiz,G.; Ramos,M.; Martin de Pozuelo,M. Rapid high-performance liquid chromatographic method for the determination of bencetonium chloride residues in fish products; confirmation by thin-layer chromatography, J.Chromatogr., 1989, 467, 321-326.

Benzocaine
Molecular formula: C9H11NO2 Molecular weight: 165.19 CAS Registry No.: 94-09-7 Merck Index: 1116 LednicerNo.: 1 9

SAMPLE

Matrix: bile, blood, tissue Sample preparation: Plasma, bile. Add three 250 |xL portions of MeOH to 250 |xL plasma and three 150 JULL portions of MeOH tol50 JULL bile, centrifuge. Remove MeOH from the supernatant using vacuum centrifugal evaporation. Dilute the residue to 1 mL with sample solvent. Filter (0.22 |xm) and inject a 100 |xL aliquot. Tissue. Homogenize l.l.g white muscle, 640 mg liver, 250 mg trunk, or 170 mg head kidneys with 5 volumes of 100 mM acetic acid. Homogenize 500 mg red muscle and 320 mg skin with 5 volumes of 100 mM and 25 mM sodium acetate at pH 4.5, respectively. Add five volumes of MeOH to the homogenates, shake horizontally for 15 min. Centrifuge at 3000 g at 15 for 15 min and remove MeOH using vacuum centrifugal evaporation. Acidify the residue with 10% acetic acid to a final acetic acid concentration of 1% (skin to 2.5% acetic acid concentration) and add to a 3 mL Cyanopropyl (Supelco) SPE cartridge, elute with MeOH. Evaporate the MeOH under nitrogen at 40. Reconstitute the residue with 500 |xL sample solvent. Filter (0.22 |xm) and inject a 100 |xL aliquot. (Sample solvent was MeOH:water:acetic acid 15: 84:1.)
HPLC VARIABLES

Guard column: 10 X 4.6 5 |xm Lichrosorb Diol Column: 150 X 4.6 3 |xm LC-18-DB Supelco Mobile phase: Gradient. A was MeOH:water:acetic acid 5:94:1. B was MeOH:water:acetic acid 55:44:1. A:B 100:0 for 3 min, to 0:100 over 5 min, maintain at 0:100 for 15 min, return to 100:0 over 1 min. Injection volume: 100 Detector: UV 286 CHROMATOGRAM Retention time: 15.5 Limit of quantitation: 1 nM OTHER SUBSTANCES Extracted: metabolites
KEYWORDS

SPE; catfish; head kidney; liver; plasma; red muscle; skin; trunk; white muscle; kidney; muscle
REFERENCE Szoke,A.; Hayton,W.L.; SchultzJ.R. Quantification of benzocaine and its metabolites in channel catfish tissues and fluids by HPLC, J.Pharm.BiomedAnaL, 1997, 16, 69-75. SAMPLE

Matrix: blood Sample preparation: Centrifuge 300 |xL whole blood at 5100 RCF for 8 min at 0. Separate plasma from erythrocytes within 30 min after withdrawing blood. Warm sample to room temperature. Vortex 130 mg (125 |xL) plasma with 250 JJLL MeOH for 1 min, centrifuge at 10 400 RCF for 15 min at 5. Filter (0.45 JJL PTFE) supernatant. Inject a 20 |mL aliquot.

HPLC VARIABLES

Guard column: 23 X 4.0 5 |mm spherical C18 with 17% carbon load, ODS type A (YMC, Inc., Wilmington) Column: 150 X 4.6 5 jjum spherical C18 with 17% carbon load, ODS type A (YMC, Inc., Wilmington) Mobile phase: MeOH: 25 mM KH2PO4 50:50 Flow rate: 1 Injection volume: 20 Detector: UV 286
CHROMATOGRAM

Retention time: 5.9 Limit of detection: 10 ng/mL Limit of quantitation: 37 ng/mL

OTHER SUBSTANCES

Extracted: ethyl p-acetamidobenzoate

KEY WORDS

plasma; trout
REFERENCE Bernardy,J.A.; Coleman,K.S.; Stehly,G.R.; Gingerich,W.H. Determination of benzocaine in rainbow trout plasma, JAOACInL, 1996, 79, 623-627. SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol:n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 |xm NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 291
CHROMATOGRAM

Retention time: 3.51 Limit of detection: <120 ng/mL

KEY WORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebuto-

lol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenqlol; aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromor amide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; dauno rubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidofiazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262.

SAMPLE Matrix: bulk Sample preparation: Prepare a 750 |xg/mL solution in 10 mM pH 2.5 orthophosphoric acid, sonicate for 10 min, filter (0.2 |xm), inject a 15 |xL aliquot.
HPLCVARIABLES

Guard column: 4 X 4 5 jjim LiChrospher 100 Column: 125 X 4 3 fim Spherisorb ODS-I Mobile phase: Gradient. A was water containing 5 mL/L 85% orthophosphoric acid and 0.56 mL/L hexylamine. B was MeCN:water 90:10 containing 5 mL/L 85% orthophosphoric acid and 0.56 mL/L hexylamine. A:B from 91:9 to 86:14 over 4 min, maintain at 86:14 for 13 min, to 55:45 over 11 min, maintain at 55:45 for 8 min, re-equilibrate at initial conditions for 20 min. Flow rate: 0.7 Injection volume: 15 Detector: UV 210 CHROMATOGRAM Retention time: 15.9
OTHER SUBSTANCES

Simultaneous: acetaminophen, acetylcodeine, caffeine, cocaine, codeine, diamorphine, lidocaine, 6-monoacety!morphine, morphine, noscapine, papaverine, procaine

REFERENCE Grogg-Sulser,K.; Helmlin,H-J-; Clerc,J.-T. Qualitative and quantitative determination of illicit heroin street samples by reversed-phase high-performance liquid chromatography: method development by CARTAGO-S, J.ChromatogrA, 1995, 692, 121-129. SAMPLE

Matrix: formulations Sample preparation: Weigh out 50 mg formulation, add 5 mL 1.5 mg/mL benzophenone in MeOH, make up to 50 mL with MeOH. Dilute 1 mL of this solution to 10 mL with MeOH, filter (0.45 fxm PTFE membrane), inject a 10 JJLL aliquot.
HPLC VARIABLES

Guard column: 30 mm long Brownlee guard column Column: 220 X 4.6 5 jxm C18 (Brownlee) Mobile phase: MeCN:water 60:40 Flow rate: 2 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 1.8 Internal standard: benzophenone (3.3) Limit of quantitation: 10000 ng/mL OTHER SUBSTANCES Simultaneous: benzyl benzoate
KEYWORDS

dermatological preparations
REFERENCE Gigante,B-; Barros,A.M.V.; Teixeira,A-; Marcelo-Curto,M.J. Separation and simultaneous high-performance liquid chromatographic determination of benzocaine and benzyl benzoate in a pharmaceutical preparation, J.Chromatogr., 1991, 549, 217-220. SAMPLE

Matrix: formulations Sample preparation: Shake 2 g powder with 10 mL EtOH for 1 h, directly inject 1 mL of this solution using a syringe-coupled nylon filter (Teknokroma).
HPLC VARIABLES

Column: 125 X 4 5 jxm Aluspher RP Select B (E. Merck) (alumina particles bonded with polybutadiene) Mobile phase: MeOH:water:diammonium phosphate 30:70:0.2 (v:v:w), pH 8.2 Flow rate: 1 Injection volume: 20 Detector: UV 191 CHROMATOGRAM Retention time: 3 OTHER SUBSTANCES Simultaneous: menthol, tyrothricin Noninterfering: gramicidin

REFERENCE Caraballo,L; Fernandez-Arevalo,M.; Holgado,M.-A.; Vela,M.-T.; Rabasco,A.-M. A rapid HPLC method for the quantification of tyrothricin, menthol, and benzocaine in pharmaceutical formulations, J.Pharm.ScL, 1994, 83, 1147-1149. SAMPLE

Matrix: formulations Sample preparation: Grind (Sorvall Omni-Mixer) and mix 14 lozenges for 2 min. Add 6.3 g powder to 15 mL mobile phase, sonicate (Bandelin Sonorex K 52) for 30 min, filter (paper), make up filtrate to 25 mL, dilute 13-fold, inject a 20 uL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 5 \im Ultrabase C18 (Scharlau) Mobile phase: MeOH: 10 mM pH 3.31 KH2PO4 75:25 Flow rate: 1 Injection volume: 20 Detector: UV 270 CHROMATOGRAM Retention time: 3.5 OTHER SUBSTANCES Simultaneous: tyrothricin Noninterfering: menthol
KEYWORDS

lozenges
REFERENCE Ortiz-Boyer,E; Tena,M.T.; Luque de Castro,M.D.; Valcarel,M. Development and validation of chromatographic methods (HPLC and GC) for the determination of the active components (benzocaine, tyrothricin and menthol) of a pharmaceutical preparation, J.Pharm.Biomed.AnaL, 1995, 13, 12971303. SAMPLE

Matrix: solutions Sample preparation: Inject a 5 |xL aliquot.

HPLC VARIABLES

Column: 300 X 4 10 jmrn jxBondapak C18 Mobile phase: MeCN:MeOH:water 20:20:60 containing 0.06% sulfuric acid, 0.5% sodium sulfate, and 0.02% sodium heptanesulfonate, pH 2.6 Flow rate: 2 Injection volume: 5 Detector: UV 305 CHROMATOGRAM Retention time: 4
OTHER SUBSTANCES

Simultaneous: butamben, lidocaine, pramoxine, procaine, tetracaine

REFERENCE Menon,G.N.; Norris,B.J. Simultaneous determination of tetracaine and its degradation product, p-nbutylaminobenzoic acid, by high-performance liquid chromatography, J.Pharm.ScL, 1981, 70, 569570.

SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 125 x 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V CHROMATOGRAM Retention time: 1.0
OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzethidine, benzoctamine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. IL Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject
a 10 JULL aliquot. HPLCVARIABLES

Column: 300 X 3.9 10 |xm ixBondapak C18 Mobile phase: MeOH:acetic acid:triethylamine:water 50:1.5:0.5:48 Flow rate: 1.5 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 10.5
OTHER SUBSTANCES

Simultaneous: butacaine, lidocaine, bupivacaine, tetracaine

REFERENCE Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 Supelcosil LC-ABZ Mobile phase: MeCN:25 mM pH 6.9 potassium phosphate buffer 35:65 Flow rate: 1.5 Injection volume: 25 Detector: UV 254 CHROMATOGRAM Retention time: 4.262
OTHER SUBSTANCES

Also analyzed: 6-acetylmorphine, amiloride, amphetamine, benzoylecgonine, caffeine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine, levorphanol, LSD, meperidine, mephobarbital, methadone, methylphenidate, methyprylon, Nnorcodeine, oxazepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE Ascah,T.L. Improved separations of alkaloid drugs and other substances of abuse using Supelcosil LCABZ column, Supelco Reporter, 1993,12(3), 18-21. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2

Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994, 18, 233-242. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 ^m LiChrospher 100 RP-8 (B)

Mobile phase: MeCN:0.025% phosphoric acidrbuffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229
CHROMATOGRAM

Retention time: 6.25 (A), 6.23 (B)

OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEY WORDS

also details of plasma extraction

REFERENCE Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology, J.ChromatogrA, 1995, 692, 103-119. SAMPLE

Matrix: sunscreen Sample preparation: Weigh out 1 g sunscreen, add 2-10 mL mobile phase, stir magnetically for 5 min, filter (0.45 jim Millex-HV), inject an aliquot.
HPLC VARIABLES

Column: 200 x 5 5 p,m Nucleosil C18 Mobile phase: MeCN: 15 mM phosphoric acid 18:82

Flow rate: 1 Injection volume: 20 Detector: UV 290 CHROMATOGRAM Limit of detection: 500 ng/mL OTHER SUBSTANCES Extracted: p-aminobenzoic acid REFERENCE Bruze,M.; Gruvberger,B.; Thulin,L PABA, benzocaine, and other PABA esters in sunscreens and aftersun products, Photodermatol.Photoimmunol.Photomed., 1990, 7, 106-108.

Benzoctamine
Molecular formula: C18H19N Molecular weight: 249.36 CAS Registry No.: 17243-39-9,10085-81-1 (HCI) Merck Index: 1117 LednicerNo.: 2 220

SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 2.3 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,

protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Benzoic acid
Molecular formula: C7H6O2 Molecular weight: 122.12 CAS Registry No.: 65-85-0 Merck Index: 1122 SAMPLE

Matrix: beverage Sample preparation: Sonicate 25 mL beverage for 15-20 min, filter (0.45 |jim) if necessary, inject a 20 (JLL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 jxm ixBondapak C18 Mobile phase: MeCN:MeOH:water:acetic acid 10:20:70:1 Flow rate: 1.5 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 12
OTHER SUBSTANCES

Simultaneous: hydroquinine, quinine, saccharin

KEYWORDS

tonic water; soft drinks

REFERENCE Valenti,L.R Liquid chromatographic determination of quinine, hydroquinine, saccharin, and sodium benzoate in quinine beverages, JAssocOffAnalChem., 1985, 68, 782-784. SAMPLE

Matrix: beverages Sample preparation: Filter sample.

HPLC VARIABLES

Column: 150 X 4.5 5 ^m Hiasil C18 (Higgins) Mobile phase: MeOH:25 mM phosphate buffer 45:55, pH 3.0 Flow rate: 1.0 Injection volume: 20 Detector: UV 218
CHROMATOGRAM

Retention time: 6.0 Limit of detection: 0.57 mg/mL OTHER SUBSTANCES Simultaneous: aspartame, caffeine
KEYWORDS

comparison with UV spectrophotometry and capillary electrophoresis; soft drinks

REFERENCE McDevitt,V.L.; Rodriguez,A.; Williams,K.R. Analysis of soft drinks: UV spectrophotometry, liquid chromatography, and capillary electrophoresis, J.Chem.Educ., 1998, 75, 625-629.

SAMPLE

Matrix: beverages, syrup Sample preparation: Dilute syrup ten fold. Filter (0.45 fxm) beverages and diluted syrup, inject a 10-20 fiL aliquot of the filtrate.
HPLC VARIABLES

Column: 300 X 3.9 10 |im ixBondapak C18 Mobile phase: MeOH:acetic acidrwater 20:5:75 Flow rate: 2 Injection volume: 10-20 Detector: UV 254 CHROMATOGRAM Retention time: 9.5 Limit of detection: 100 ng
OTHER SUBSTANCES

Simultaneous: acesulfame, caffeine, dulcin, p-hydroxybenzoic acid, saccharin, vanillin

REFERENCE Veerabhadrarao,M.; Narayan,M.S.; Kapur,O.; Sastry,C.S. Reverse phase liquid chromatographic determination of some food additives, JAssoc.Off.Anal.Chem., 1987, 70, 578-582. SAMPLE

Matrix: bile, blood Sample preparation: Blood, plasma. 250 |xL Blood or plasma + 50 JJLL 16 |xg/mL methoxybenzoic acid in water + 800 fxL MeCN, mix, centrifuge. Remove the supernatant and dry it under a stream of nitrogen, reconstitute the residue in 200 |xL MeCN:0.5% acetic acid 10:90, centrifuge, inject an aliquot. Bile. Inject bile directly.
HPLC VARIABLES

Guard column: 22 X 3.4 37-50 |xm C18 (Waters) Column: 300 X 3.9 10 |xm Ultrasphere Mobile phase: Gradient. MeCN:0.5% acetic acid 10:90 for 10 min then to 72.5:27.5 over 2 min, maintain at 72.5:27.5 for 8 min, return to initial conditions over 1 min, re-equilibrate for 4 min. Flow rate: 1 Detector: UV 254
CHROMATOGRAM

Retention time: 17.8 Internal standard: methoxybenzoic acid (19.1)

OTHER SUBSTANCES

Extracted: hippuric acid, benzoyl glucuronide

KEYWORDS

plasma
REFERENCE Chiba,M.; Poon,K.; Hollands,J.; Pang,K.S. Glycine conjugation activity of benzoic acid and its acinar localization in the perfused rat liver, J.Pharmacol.Exp.Ther., 1994, 268, 409-416. SAMPLE Matrix: blood

Sample preparation: Condition a 100 mg Bond Elut C18 SPE cartridge with 2 mL MeCN and 2 mL pH 3 aqueous acetic acid. Dilute I m L serum with 1 mL pH 3 aqueous acetic

acid, add to SPE cartridge, wash with 500 |xL pH 3 aqueous acetic acid, elute with 1 mL MeCN acidified to pH 2.5 with acetic acid, inject a 20 jxL aliquot of the eluate.
HPLC VARIABLES

Column: Capcell Pak C-18 AG-120 (Shiseido) Mobile phase: MeCN:water 20:80 Flow rate: 1.2 Injection volume: 20 Detector: UV 250
CHROMATOGRAM Retention time: 5.8 KEYWORDS

serum; dental material; methyl methacrylate polymer; plexiglass; horse; SPE

REFERENCE Shintani,H.; Tsuchiya,T.; Hata,Y.; Nakamura,A. Solid phase extraction and HPLC analysis of toxic components eluted from methyl methacrylate dental materials, J.Anal.ToxicoL, 1993, 17, 73-78. SAMPLE

Matrix: bulk, formulations Sample preparation: 100 mg Bulk drug or formulation containing 100-120 mg drug + 10 mL 1 (bulk) or 4 (formulations) mg/mL benzoic acid in MeOH:water 50:50 + 20 mL 5 mg/ mL p-nitroacetophenone in MeOH, make up to 100 mL with water, inject an aliquot.
HPLC VARIABLES

Column: 300 X 4 10 \xm ixBondapak C18 Mobile phase: MeCN:MeOH:water:glacial acetic acid 20:5:74:1 containing 0.05-0.08% sodium 1-heptanesulfonate, pH 3.1 Flow rate: 2 Injection volume: 5 Detector: UV 278
CHROMATOGRAM

Retention time: 6 Internal standard: benzoic acid, p-nitroacetophenone (12)

OTHER SUBSTANCES

Simultaneous: 4-amino-2-chlorobenzoic acid, impurities, chloroprocaine

KEYWORDS

benzoic acid is IS
REFERENCE Menon,G.; Norris,B.; Webster,J. Simultaneous determination of chloroprocaine hydrochloride and its degradation product 4-amino-2-chlorobenzoic acid in bulk drug and injection solutions by high-performance liquid chromatography, J.Pharm.ScL, 1984, 73, 251-253. SAMPLE

Matrix: food Sample preparation: 5 g Food + 2 mL 1 mg/mL 4-hydroxyacetanilide in MeOH, make up to 15 mL with MeOH, mix, centrifuge at 1500 g for 10 min, filter an aliquot of the supernatant (0.45 |xm), inject a 15 jxL aliquot.
HPLC VARIABLES

Guard column: RP18 (Brownlee)

Column: 10 |xm Spheri RP-18 (Brownlee) Mobile phase: MeOH:30 mM pH 6.5 phosphate buffer 5:95 Flow rate: 2 Injection volume: 15 Detector: UV 227
CHROMATOGRAM

Retention time: 4.24 Internal standard: 4-hydroxyacetanilide (14.57) OTHER SUBSTANCES Simultaneous: sorbic acid
KEYWORDS

beverages; fruit; seafood; vegetables; sauces; dairy products

REFERENCE Bui,L.V.; Cooper,C. Reverse-phase liquid chromatographic determination of benzoic and sorbic acids in foods, JAssoc.OffAnalChem., 1987, 70, 892-896. SAMPLE

Matrix: food
Sample preparation: 2 g Soy sauce or 1 g sugared fruit or roast beef + 1 O g NaCl, make up to 50 mL with acetone, swirl vigorously, let stand for 30 min, filter, wash the solid with acetone, make up filtrate to 50 mL with acetone, inject a 20 |xL aliquot. HPLC VARIABLES

Column: 250 X 4.6 5 jutm monomeric C18 (Shoko, Kyoto) Mobile phase: MeCN:50 mM pH 4.5 a-hydroxyisobutyric acid in water 22:34 containing 2.5 mM hexadecyltrimethylammonium bromide Flow rate: 1 Injection volume: 20 Detector: UV 233 CHROMATOGRAM Retention time: 24
OTHER SUBSTANCES

Simultaneous: acesulfame-K, 3-t-butyl-4-hydroxyanisole, butyl p-hydroxybenzoate, t-butylhydroxyquinone, dulcin, ethyl p-hydroxybenzoate, isobutyl p-hydroxybenzoate, isopropyl p-hydroxybenzoate, methyl p-hydroxybenzoate, saccharin, sodium dehydroacetate, sorbic acid
KEYWORDS

soy sauce; roast beef; sugared fruit

REFERENCE Chen,B.H.; Fu,S.C. Comparison of extraction methods and column types for the determination of additives by liquid chromatography, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 625-643. SAMPLE

Matrix: formulations Sample preparation: 5 mL Formulation + 5 mL IS solution, make up to 50 mL with MeOH, inject 10 JJLL aliquot. (IS solution was 0.2 mg p-nitroacetophenone and 2.5 mg isobutyrophenone per mL of MeOH.)

HPLC VARIABLES

Column: 300 X 4 10 |xm jxBondapak C18 Mobile phase: MeCN:water:reagent 25:60:15, pH 2.6 (Reagent was MeOH containing 0.06% sulfuric acid, 0.5% sodium sulfate, and 0.02% sodium heptanesulfonate.) Flow rate: 2 Injection volume: 10 Detector: UV 257
CHROMATOGRAM

Retention time: 3.5 Internal standard: p-nitroacetophenone (6) and isobutyrophenone (13)
OTHER SUBSTANCES

Simultaneous: benzyl alcohol, hydroxyzine, benzaldehyde, p-chlorobenzoic acid, p-chlorobenzaldehyde, p-chlorobenzophenone

KEY WORDS

injections; stability-indicating
REFERENCE Menon,G.N.; Norris,B.J. Simultaneous determination of hydroxyzine hydrochloride and benzyl alcohol in injection solutions by high-performance liquid chromatography, J.Pharm.ScL, 1981, 70, 697-698. SAMPLE

Matrix: formulations Sample preparation: Leach 200 or 300 mg ground capsule or tablet with water or mobile phase and dilute to 50 mL, sonicate for 5 min, centrifuge at 2500 rpm for 5 min, inject an aliquot. Dilute 4-25 mL of liquid formulations to 250 mL with water, inject an aliquot.
HPLC VARIABLES

Column: Partisil-10 C8 Mobile phase: MeOH:MeCN:water:PIC-B5 50:170:755:25 (PIC-B5 (Waters) is 200 mM sodium pentanesulfonate in glacial acetic acid.) Flow rate: 2 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 14.5
OTHER SUBSTANCES

Simultaneous: phenylephrine, phenylpropanolamine, guaifenesin, impurities, degradation products

KEY WORDS

tablets; capsules; liquid formulations; stability-indicating

REFERENCE Schieffer,G.W.; Smith,W.O.; Lubey,G.S.; Newby,D.G. Determination of the structure of a synthetic impurity in guaifenesin: modification of a high-performance liquid chromatographic method for phenylephrine hydrochloride, phenylpropanolamine hydrochloride, guaifenesin, and sodium benzoate in dosage forms, J.Pharm.ScL, 1984, 73, 1856-1858. SAMPLE

Matrix: formulations

HPLC VARIABLES

Column: 300 X 3.9 jiBondapak C18 Mobile phase: MeOH:buffer 20:80 (Buffer was 15 mM 1-butanesulfonic acid + 15 mM KH2PO4 + 2 mIVL triethylamine, pH adjusted to 4.8 0.1 with dilute phosphoric acid.) Column temperature: 50 Flow rate: 2 Injection volume: 20 Detector: UV 214
CHROMATOGRAM Retention time: 3.9 OTHER SUBSTANCES

Simultaneous: codeine, acetaminophen, p-aminophenol, codeine N-oxide, codeinone

KEY WORDS

elixir; stability-indicating
REFERENCE Sisco,W.R.; Rittenhouse,C.T.; Everhart,L.A.; McLaughlin,A.M. Simultaneous high-performance liquid chromatographic stability-indicating analysis of acetaminophen, codeine phosphate, and sodium benzoate in elixirs, J.Chromatogr., 1986, 354, 355-366. SAMPLE

Matrix: formulations Sample preparation: Weigh out 2 g metronidazole benzoate suspension containing 3.5% metronidazole benzoate, add 80 mL MeOH:water 80:20, sonicate for a few minutes, make up to 100 mL with MeOH water 80:20, centrifuge at 900 g for 10 min, inject a 10 |JLL aliquot.
HPLC VARIABLES

Guard column: 50 X 2 30-38 |xm Whatman Co:Pell Column: 250 X 4.6 10 |xm Perkin-Elmer C18 Mobile phase: MeOH:water 60:40 containing 5 mM acetate buffer and 50 mM KNO3, pcH* 5.2 Flow rate: 1 Injection volume: 10 Detector: UV 254
CHROMATOGRAM Retention time: 6 OTHER SUBSTANCES

Simultaneous: metronidazole, metronidazole benzoate, methylparaben, propylparaben

KEY WORDS

suspensions
REFERENCE PashankoVjP.R; Kostova,L.L. Reversed-phase high-performance liquid chromatography of metronidazole benzoate in suspension dosage form, J.Chromatogr., 1987, 394, 382-387. SAMPLE

Matrix: formulations Sample preparation: Dilute 1 mL syrup to 50 mL with mobile phase, filter (0.45 |xm), inject 20 u-L aliquot.

HPLC VARIABLES

Column: 250 X 4.6 Zorbax CN Mobile phase: MeCN:water:formic acid:methanesulfonic acid 500:500:1:1, pH adjusted to 3.5 with 10% NaOH Flow rate: 1 Injection volume: 20 Detector: UV 290
CHROMATOGRAM Retention time: 3 OTHER SUBSTANCES

Simultaneous: guaifenesin, saccharin, dextromethorphan

KEY WORDS

syrup
REFERENCE Chen,T.M.; Pacifico,J.R.; DaIy5R-E. High-pressure liquid chromatographic assay of dextromethorphan hydrobromide, guaifenesin, and sodium benzoate in an expectorant syrup, J.Chromatogr.ScL, 1988, 26, 636-639. SAMPLE

Matrix: formulations Sample preparation: Weigh out ground tablets or capsules equivalent to 150 mg bepridil.HCl, add 150 mL MeCN, shake for 30 min, dilute to 200 mL with MeCN, filter (Schleicher & Schiill paper, grade 588), inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 300 X 4.6 10 urn uBondapak C18 Mobile phase: MeCNrbuffer 580:405 (Buffer was 1.1 g sodium 1-heptanesulfonate in 405 mL water, adjust to pH 2.37 with glacial acetic acid (ca. 15 mL).) Column temperature: 35 Flow rate: 1.3 Injection volume: 20 Detector: UV 254
CHROMATOGRAM Retention time: 2.6 OTHER SUBSTANCES

Simultaneous: impurities, benzaldehyde, N-benzylaniline, bepridil

KEY WORDS

capsules; tablets
REFERENCE Renzi,N.L.; Fronheiser,M.E.; Duong,H.T.; Fulton,D.J.; Rabinowitz,M. Stability-indicating high-performance liquid chromatography assay for bepridil hydrochloride drug substance and drug products, J.Chromatogr., 1989, 462, 398-405. SAMPLE

Matrix: formulations
HPLC VARIABLES Column: C18

Mobile phase: MeCNrbuffer 13:87 (Buffer was 0.01% acetic acid containing 0.001% sodium octanesulfonate.) Flow rate: 2.5 Detector: UV 280
CHROMATOGRAM

Internal standard: benzoic acid OTHER SUBSTANCES Simultaneous: dopamine

KEYWORDS

injections; 5% dextrose; benzoic acid is IS

REFERENCE Pramar,Y.; Das Gupta,V.; Gardner,S.N.; Yau,B. Stabilities of dobutamine, dopamine, nitroglycerin and sodium nitroprusside in disposable plastic syringes, J.Clin.Pharm.Ther., 1991, 16, 203-207. SAMPLE

Matrix: formulations Sample preparation: Dilute 10 mL to 1 L with water.

HPLC VARIABLES

Column: 300 X 3.9 10 |xm ixBondapak C18 Mobile phase: MeCN:water:diethylamine:glacial acetic acid 250:739:1:10, apparent pH 4.1 Column temperature: 35 Flow rate: 1.3 Injection volume: 25 Detector: UV 273 CHROMATOGRAM Retention time: 5.3
OTHER SUBSTANCES

Simultaneous: guaifenesin, dextromethorphan

KEY WORDS

liquid formulations; stability-indicating

REFERENCE Wilson,T.D.; Jump,W.G.; Neumann,W.C.; San Martin,T. Validation of improved methods for high-performance liquid chromatographic determination of phenylpropanolamine, dextromethorphan, guaifenesin and sodium benzoate in a cough-cold formulation, J.Chromatogr., 1993, 641, 241-248. SAMPLE

Matrix: formulations Sample preparation: Weigh out 4 mL of 1 mg/mL solution and make up to 100 mL with mobile phase, inject 20 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 Zorbax cyano special Mobile phase: MeCN:buffer 50:50 (Buffer was 1% triethylamine, pH adjusted to 6 with cone phosphoric acid.) Flow rate: 1 Injection volume: 20 Detector: UV 215

CHROMATOGRAM Retention time: 4 OTHER SUBSTANCES Simultaneous: fluoxetine KEY WORDS syrup; elixir REFERENCE Peterson,J.A.; Risley,D.S.; Anderson,P.N.; Hostettler,K.F. Stability of fluoxetine hydrochloride in fluoxetine solution diluted with common pharmaceutical diluents, Am.J.Hosp.Pharm., 1994, 51, 13421345. SAMPLE Matrix: juice Sample preparation: Condition a Sep-Pak Classic SPE cartridge with 2 mL MeOH and 5 mL water. Centrifuge 10 mL orange juice at 1500 g for 5 min, add 1 mL supernatant to the SPE cartridge, wash with 3 mL hexane.MeCN 98:2, force air through cartridge three times, elute with 3 mL MeOH, make eluate up to 3 mL with MeOH, filter (0.45 |xm) HPLC VARIABLES Guard column: PRP-I (Hamilton) Column: 250 X 4.1 10 |xm PRP-I (Hamilton) Mobile phase: MeCN:buffer 40:60 (Buffer was 6.8 g KH2PO4 in 1 L water, adjust pH to 2.3 with 85% phosphoric acid.) Flow rate: 0.7 Injection volume: 10 Detector: UV 230 CHROMATOGRAM Retention time: 7.26 Limit of detection: 0.5 ppm KEYWORDS orange; SPE REFERENCE Lee,H.S. Liquid chromatographic determination of benzoic acid in orange juice: Interlaboratory study, JAOAC Int., 1995, 78, 80-82. SAMPLE Matrix: perfusate HPLC VARIABLES Column: Lichrospher 100 RP-18 Mobile phase: MeOH:water containing 10 mM citric acid 45:55 Column temperature: 45 Flow rate: 1 Detector: UV 230 KEY WORDS rat; intestine; Caco-2 monolayer REFERENCE Takagi,M.; Taki,Y; Sakane,T.; Nadai,T.; Sezaki,H.; Oku,N.; Yamashita,S. A new interpretation of salicylic acid transport across the lipid bilayer: Implication of pH-dependent but not carrier-mediated absorption from the gastrointestinal tract, J.Pharmacol.Exp.Ther., 1998, 285, 1175-1180.

SAMPLE

Matrix: solutions Sample preparation: Add 500 |JLL of a solution in MeCN to 100 mg finely powdered potassium carbonate, add 250 |xL 3.8 mM 18-crown-6 in MeCN, add 250 fxL 0.8 mM reagent in MeCN, heat at 80 in the dark for 20 min, cool, inject a 5 |xL aliquot. (Synthesize the reagent, 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone, as follows. Stir 483 g veratrole in 1.45 L acetic acid at 15 for 1 h, add 683 g concentrated nitric acid (d 1.05) over 1 h (maintain the temperature below 40 by cooling and regulating the rate of addition of the nitric acid). Continue stirring and add 2.127 L fuming nitric acid (d 1.50) over 1 h while maintaining the temperature below 30, let stand for 2 h, pour into a large volume of cold water, filter, wash the solid with water until the washings are neutral, recrystallize from EtOH to give 4,5-dinitroveratrole (mp 129.5-130.5) (J. Am. Chem. Soc. 1946, 68, 1536). Reflux 5 g 4,5-dinitroveratrole in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh iron powder and 20 mL concentrated HCl in small portions over 1 h, reflux for 4 h, add 10 mL water, reflux for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with 200 mL portions of benzene. Combine the organic layers and evaporate them to dryness, add 10 mL concentrated HCl, recrystallize from EtOH to give l,2-diamino-4,5-dimethoxybenzene monohydrochloride as very slightly pink needles (mp 240) (Anal. Chim. Acta 1982, 134, 39). Heat 2.5 mmoles l,2-diamino-4,5dimethoxybenzene hydrochloride and 2.4 mmoles pyruvic acid in 30 mL 500 mM HCl on a boiling water bath for 2 h, cool with ice-water, filter. Wash the precipitate with water and dry it under vacuum, recrystallize from MeOHrwater 90:10 to give 6,7-dimethoxy-3methyl-2(lH)-quinoxalinone as yellow needles (mp 255) (Chem. Pharm. Bull. 1985, 33, 3493). Treat 1 g 6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone dissolved in 50 mL anhydrous MeOH with a solution of diazomethane in ether, evaporate to dryness under reduced pressure, dissolve the residue in 5 mL ethyl acetate, chromatograph on a 250 X 35 column filled with 130 g 70-230 mesh silica gel 60 (Merck) using n-hexane:ethyl acetate 25:75 to give 6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone as yellow needles (mp 170171). Dissolve 350 mg 6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone in 3 mL acetic acid, add 350 mg anhydrous sodium acetate, add 2 mL 1.5 M bromine in acetic acid, heat at 100 for 15 min, cool, add 10 mL ether, filter, wash the solid 2 or 3 times with small portions of ether. Combine the filtrate and washings and evaporate them to dryness, dissolve the residue in 5 mL ethyl acetate, chromatograph on a 250 X 35 column filled with 130 g 70-230 mesh silica gel 60 (Merck) using ether, evaporate the main fraction to dryness, recrystallize the residue from n-hexane:ethyl acetate 50:50 to give 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone as yellow needles (mp 161-163).)
HPLC VARIABLES

Column: 100 X 4 10 (xm Radial-Pak C18 (Waters) Mobile phase: Gradient. MeOH:water from 57:43 to 100:0 over 20 min, maintain at 100:0 for 12 min Flow rate: 2 Injection volume: 5 Detector: F ex 370 em 450
CHROMATOGRAM

Retention time: 9.4 Limit of detection: 0.3-1 fmole

OTHER SUBSTANCES

Simultaneous: p-aminobenzoic acid, arachidic acid, arachidonic acid, butyric acid, capric acid, caproic acid, caprylic acid, deoxyuridine, glucuronic acid, imidazole-4-acetic acid, lauric acid, linoleic acid, linolenic acid, margaric acid, l-methyl-4-imidazoleacetic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, propionic acid, salicylic acid, stearic acid, thymidine, uridine, valeric acid
KEY WORDS

derivatization

REFERENCE Yamaguchi,M.; Hara,S.; Matsunaga,R.; Nakamura,M.; Ohkura,Y. 3-Bromomethyl-6,7-dimethoxy-lmethyl-2(lH)-quinoxalinone as a new fluorescence derivatization reagent for carboxylic acids in highperformance liquid chromatography, J.Chromatogr., 1985, 346, 227-236. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject a 10 jxL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 10 |xm jxBondapak C18 Mobile phase: MeOH:acetic acid:triethylamine:water 40:1.5:0.5:58 Flow rate: 1.5 Injection volume: 10 Detector: UV 261
CHROMATOGRAM Retention time: 9 OTHER SUBSTANCES

Simultaneous: ascorbic acid, salicylic acid, quinine, dihydroquinine

REFERENCE Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE

Matrix: solutions Sample preparation: Prepare an aqueous solution, inject a 10 jxL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 ujn Nucleosil C18 Mobile phase: MeCN:MeOH:buffer:triethylamine 4:4:92:0.01 (Buffer was 0.05% sodium octanesulfonate adjusted to pH 2.2 with 3 M phosphoric acid.) Flow rate: 1.5 Injection volume: 10 Detector: UV 215
CHROMATOGRAM Retention time: 2.4 OTHER SUBSTANCES

Simultaneous: p-aminobenzoic acid, benzaldehyde, benzyl alcohol, protirelin

REFERENCE Rao,G.N.; Sutherland,J.W.; Menon,G.N. High-performance liquid chromatographic assay for thyrotropin releasing hormone and benzyl alcohol in injectable formulation, Pharm.Res., 1987, 4, 38-41. SAMPLE

Matrix: solutions Sample preparation: React the carboxylic acid, triethylamine, and l-(2,5-dihydroxyphenyl)-2-bromoethanone in a 1:2:4 molar ratio in MeCN at 45 for 2 h, inject a 10 |xL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bromoethanone is as follows. Stir 27.6 g 1,4-dimethoxybenzene and 28 mL bromoacetyl bromide at 0, add 53.4 g aluminum bromide over 10 min (an exothermic reactions ensues), let stand at room temperature for

12 h, add 100 mL 48% HBr, add 100 g ice, stir for 1 h, extract twice with 200 mL portions of diethyl ether. Combine the extracts and wash them 3 times with 200 mL portions of water, dry over 40 g anhydrous magnesium sulfate, evaporate to dryness, recrystallize the product 3 times from EtOH to yield l-(2,5-dihydroxyphenyl)-2-bromoethanone monobromoacetate (mp 105-107). Dissolve H g l-(2,5-dihydroxyphenyl)-2-bromoethanone monobromoacetate in 200 mL warm dry MeOH saturated with HBr, stir for 18 h, add 200 mL water, cool to -10. Collect the yellow solid and dry it under vacuum at 50 for 48 h, recrystallize from toluenerheptane 50:50 then toluene to obtain l-(2,5-dihydroxyphenyl)-2-bromoethanone as yellow needles (mp 117-119).)
HPLC VARIABLES

Column: 250 X 4 7 |xm RP-18 LiChrocart (Merck) Mobile phase: MeOH: 100 mM pH 6.5 sodium acetate 58:42 Flow rate: 1 Injection volume: 10 Detector: E, Bioanalytical Systems Model LC4B, glassy carbon electrode 0.6 V, Ag/AgCl reference electrode
CHROMATOGRAM

Retention time: 5 Limit of detection: 1 pmole

OTHER SUBSTANCES

Simultaneous: quinoxaline-2-carboxylic acid, salicylic acid

KEYWORDS

derivatization
REFERENCE Munns,RK; Roybal,J.E.; Shimoda,W.; Hurlbut,J.A. l-(4-Hydroxyphenyl)-, l-(2,4-dihydroxyphenyl)-and l-(2,5-dihydroxyphenyl)-2-bromoethanones: new labels for determination of carboxylic acids by highperformance liquid chromatography with electrochemical and ultraviolet detection, J.Chromatogr., 1988, 442, 209-218. SAMPLE

Matrix: solutions Sample preparation: Inject 50 |xL onto column A in series with column B, after 1.1 min switch so that column A comes after column B, continue to elute.
HPLC VARIABLES

Column: A 15 X 3.2 7 |xm New Guard RP-18 (Applied Biosystems); B 100 X 4.6 3 jmi Econosphere C18 (Alltech) Mobile phase: MeCN:water:phosphoric acid 25:75:0.2 Flow rate: 1 Injection volume: 50 Detector: UV 200
CHROMATOGRAM

Retention time: 6 Limit of detection: 0.05 ppm OTHER SUBSTANCES Extracted: toluene, cresol, phenol
KEYWORDS

groundwater; water; column-switching

REFERENCE Chamkasem,N.; Hill,K.D.; Sewell,G.W. High-performance liquid chromatographic column-switching technique for the determination of intermediates of anaerobic degradation of toluene in ground water microcosm, J.Chromatogr., 1991, 587, 185-191. SAMPLE

Matrix: solutions Sample preparation: Mix a 100 |L aliquot of a 5-1000 nM solution of a carboxylic acid in MeCN with 100 |xL 18-crown-6 solution, add 100 |xL 100 u-M dansyl-BAP in MeCN, mix, let stand at room temperature (aliphatic acids) or 55 (aromatic acids) for 30 min, add 100 u.L 3 mM thymine in MeCN, add 5 mg potassium bicarbonate, vortex for 30 s, let stand for 30 min, evaporate to dryness under a stream of nitrogen. Reconstitute with dichloromethane, add to a Bond-Elut silica SPE cartridge, elute with 1.5 mL MeCN:dichloromethane 50:50. Evaporate the eluate to dryness and reconstitute the residue with 500 |xL mobile phase, inject a 25 |JLL aliquot. (Prepare 18-crown-6 solution by sonicating a 1 mg/mL solution of 18-crown-6 in MeCN containing 1 mg/mL potassium bicarbonate for 20 min. Prepare dansyl-BAP (N-(bromoacetyl)-N>-[5-(dimethylamino)naphthalene-lsulfonyl]piperazine) as follows. Slowly add a solution of 135 mg dansyl chloride in 30 mL acetone to a 10-fold molar excess of piperazine in acetone:water 75:25, stir at 50 for 1 h, evaporate the acetone. Acidify the remaining aqueous layer with concentrated nitric acid, wash 3 times with 15 mL portions of dichloromethane, adjust the pH of the aqueous layer to 11 with concentrated NaOH, extract three times with 15 mL portions of dichloromethane. Combine the extracts and dry them over anhydrous calcium chloride, concentrate to about 5 mL, chromatograph on a 400 X 25 column of 60-200 jxm silica gel Si-60, wash with about 20 mL dichloromethane:MeOH 99:1 to remove a small fluorescent band, elute with about 30 mL dichloromethane:MeOH 95:5-94:6 to obtain a solution of dansylpiperazine, determine the concentration by UV absorption at 340 nm (extinction coefficient = 4300 in MeOH). Stir a solution of 700 mg bromoacetic acid and 1.1 g dicyclohexylcarbodiimide in 100 mL MeCN at room temperature for 1 min, slowly add 470 jimoles dansylpiperazine in MeCN, stir for 1 h, evaporate to dryness, reconstitute the residue with 10 mL dichloromethane, filter. Chromatograph the filtrate on a 400 X 25 column of 40-60 |xm Si-60 silica gel with dichloromethane, when the first-eluting, strongly-fluorescent yellow band reaches the outlet change the eluent to dichloromethane:MeOH 99:1, collect about 50 mL eluate to obtain dansyl-BAP.)
HPLC VARIABLES

Column: 150 X 3.1 5 jim LiChrosorb RP-18 Mobile phase: MeCN:water 60:40, containing 2.5 mM pH 7.0 imidazole buffer

Flow rate: 0.5 Injection volume: 25

Detector: F ex 246 em 490 (cut-off filter) following post-column reaction. The column effluent mixed with 50 mM hydrogen peroxide in MeCN containing 5 mM bis(2-nitrophenyl)oxalate pumped at 0.3 mL/min and the mixture flowed immediately to the detector. (Prepare bis(2-nitrophenyl)oxalate by dissolving 13.9 g 2-nitrophenol in 250 mL benzene (Caution! Benzene is a carcinogen!), remove 50 mL benzene by azeotropic distillation, cool to 10, add 10.1 g freshly distilled triethylamine, add 7 g oxalyl chloride dropwise, allow to warm to room temperature, let stand overnight, evaporate to dryness under reduced pressure, recrystallize to give bis(2-nitrophenyl)oxalate (J. Chem. Educ. 1974, 51, 529).)
CHROMATOGRAM

Retention time: 24 Limit of detection: 0.8-1 pmole

OTHER SUBSTANCES

Simultaneous: 2,4-dichlorobenzoic acid, ibuprofen, lipoic acid, 2-methoxybenzoic acid, naproxen, octanoic acid

KEYWORDS

derivatization; post-column reaction; SPE

REFERENCE Kwakman,P.J.M.; Van Schaik,H.R; Brinkman,U.A.T.; de Jong,G.J. iV-(Bromoacetyl)-iNT-[5-(dimethylamino)naphthalene-l-sulfonyl]piperazine as a sensitive labeling reagent for the determination of carboxylic acids by liquid chromatography with peroxyoxalate chemiluminescence and fluorescence detection, Analyst, 1991, 116, 1385-1391. SAMPLE

Matrix: solutions Sample preparation: Dilute a 10 mg/mL solution of caffeine in water 1:1000 with mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 2540 X 4.6 5 |xm C18 (Supelco) Mobile phase: MeOH: 10 mM ammonium acetate and 2.5 mM sodium heptanesulfonate 20: 80, pH adjusted to 5.1 with glacial acetic acid Flow rate: 2 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 8 OTHER SUBSTANCES Simultaneous: caffeine
KEYWORDS

water
REFERENCE Donnelly,R.R; Tirona,R.G. Stability of citrated caffeine injectable solution in glass vials, Am.J.Hosp.Pharm., 1994, 51, 512-514. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cor-

tisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994, 18, 233-242. SAMPLE

Matrix: solutions Sample preparation: Prepare a 1-10 |xg/mL solution in water, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jjim Hypersil SCX/C18 Mobile phase: MeCN:25 mM pH 3 Na2HPO4 50:50 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: k' 0.69

OTHER SUBSTANCES

Also analyzed: amitriptyline, barbital, butabarbital, clomipramine, clonazepam, desipramine, diazepam, flurazepam, furosemide, imipramine, nitrazepam, phenobarbital, phenol, phenolphthalein, pindolol, propranolol, resorcinol, salicylic acid, secobarbital, terbutaline, xylazine
KEYWORDS

effect of mobile phase pH on capacity factor is discussed

REFERENCE Walshe,M.; Kelly,M.T.; Smyth,M.R.; Ritchie,H. Retention studies on mixed-mode columns in high-performance liquid chromatography, J.ChromatognA, 1995, 708, 31-40. SAMPLE

Matrix: solutions Sample preparation: Acidify 1 mL solution with 200 |xL 100 mM HCl, extract with ether. Evaporate the ether to dryness, reconstitute with 3 mL mobile phase, inject a 20 |JLL aliquot.
HPLC VARIABLES

Guard column: 4 x 4 Lichrosphere 100RP-18 Column: 250 X 4 Lichrosphere 100RP-18 Mobile phase: MeCN: 10 mM pH 6.8 phosphate buffer containing 10 mM sec-butylamine 5:95 Column temperature: 40 Flow rate: 1 Injection volume: 20 Detector: F ex 305 em 407 CHROMATOGRAM Retention time: 4.5 OTHER SUBSTANCES Simultaneous: 4-aminosalicylic acid
REFERENCE Motohashi,N.; Saito,Y. Rate constants for reaction of hydroxyl radicals with sulfapyridine and aminosalicylic acids, Chem.Pharm.Bull., 1996, 44, 163-166. SAMPLE

Matrix: solutions Sample preparation: Dissolve salt of pindolol in MeOHrwater 50:50, inject an aliquot.
HPLC VARIABLES

Column: 200 X 4.6 5 jxm Hypersil RP-18 Mobile phase: Gradient. MeOH:buffer from 20:80 to 80:20 over 10 min. (Buffer was 2% acetic acid containing 1.1% sodium 1-heptanesulfonate.) Column temperature: 40 Flow rate: 1.5 Detector: UV 273 CHROMATOGRAM Retention time: 5.9
OTHER SUBSTANCES

Simultaneous: 2-methoxyphenylacetic acid (UV 270), pindolol (UV 254)

REFERENCE Pietilainen,H-; Saesmaa,T. HPLC determination of pindolol benzoate and pindolol 2-methoxyphenylacetate, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 583-591.

SAMPLE Matrix: urine Sample preparation: Centrifuge urine at 2000 g for 10 min, filter (0.45 |xm Millex-HV), dilute ten-fold with water, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 150 X 3.9 4 jjurn Nova-Pak C18 Mobile phase: MeOH:25 mM pH 4.5 acetate buffer 5:95 Column temperature: 35 Flow rate: 1 Injection volume: 20 Detector: UV 230
CHROMATOGRAM

Retention time: 10.4 Limit of detection: 1000 ng/mL

OTHER SUBSTANCES

Extracted: phenylacetic acid, hippuric acid, phenaceturic acid

KEYWORDS

sheep
REFERENCE Arin,M.J.; Diez,M.T.; Resines,J.A. Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1992, 582, 13-18. SAMPLE

Matrix: vegetables Sample preparation: Place 25 g homogenized vegetables in a flask and make up to 100 mL with MeOH:water 60:40, shake vigorously for 10 min, filter (paper). Centrifuge an aliquot of the filtrate at 11600 g for 10 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES

Guard column: 10 X 4 Spherisorb ODS-2 Column: 250 X 4 Spherisorb ODS-2 Mobile phase: 30 mM pH 6.7 Phosphate buffer (Buffer was 2.5 g K2HPO4.3H2O and 2.5 g KH2PO4 in 1 L water.) Flow rate: 2 Injection volume: 20 Detector: UV 230 CHROMATOGRAM Retention time: 2.5 Limit of detection: 1 ppm OTHER SUBSTANCES Extracted: sorbic acid
KEY WORDS

peppers; tomatoes; caperberries; onions

REFERENCE Montafio,A.; Sanchez,A.H.; Rejano,L. Determination of benzoic and sorbic acids in packaged vegetable products. Comparative evaluation of methods, Analyst, 1995, 120, 2483-2487. SAMPLE

Matrix: wine Sample preparation: Adjust pH of wine to 7-8 with potassium bicarbonate. Remove a 1 mL aliquot and add it to 1 mL 170 mM phenacyl bromide in acetone, add 1 mL 17 mM 18-crown-6 in acetone, add 1 mL acetone, heat in a boiling water bath for 75 min, cool, inject a 10 |xL aliquot. (Recrystallize phenacyl bromide from n-heptane.)
HPLC VARIABLES

Guard column: 37-50 |xm Bondapak C18/Corasil Column: 250 X 4 7 jim RP-18 (Merck) Mobile phase: Gradient. MeOH:water from 35:65 to 85:15 over 20 min. Flow rate: 2 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: 14.7
OTHER SUBSTANCES

Extracted: acetic acid, anisic acid, benzilic acid, butyric acid, caprylic acid, cinnamic acid, citramalic acid, citric acid, enanthic acid, fumaric acid, galacturonic acid, gallic acid, glutaric acid, glycolic acid, glyoxylic acid, p-hydroxybenzoic acid, isocitric acid, a-ketoglutaric acid, lactic acid, malic acid, mandelic acid, phenylacetic acid, propionic acid, protocatechuic acid, pyruvic acid, salicylic acid, sorbic acid, succinic acid, tartaric acid, valeric acid, vanillic acid, ascorbic acid
KEY WORDS

derivatization
REFERENCE Mentasti,E.; Gennaro,M.C; Sarzanini,C; Baiocchi,C; Savigliano,M. Derivatization, identification and separation of carboxylic acids in wines and beverages by high-performance liquid chromatography, J.Chromatogr., 1985, 322, 177-189. SAMPLE

Matrix: yogurt Sample preparation: 500 mg Homogenized yogurt + 7 mL buffer, sonicate for 2 min, shake mechanically for 20 min, centrifuge at 2000 rpm for 10 min, repeat extraction twice more. Combine the supernatants and make up to 25 mL with buffer. Remove a 5 mL extract and add it to 5 mL 10 mM tri-n-octylamine in chloroform, shake for 20 min, centrifuge at 2000 rpm for 10 min. Remove 2.5 mL of the organic phase and add it to 2.5 mL 100 mM sodium perchlorate in water, extract, centrifuge, inject an aliquot of the aqueous phase. (Buffer was 24.650 g NaH2PO4.H2O and 1.260 g Na2HPO4.2H2O made up to 2 L with water, pH 5.5.)
HPLC VARIABLES

Column: 250 X 4 10 |xm RP-18 (Merck) Mobile phase: MeOH:buffer 40:60 (Buffer was 900 (xL 1 M phosphoric acid and 27.598 g NaH2PO4.H2O made up to 2 L with water, pH 4.5.) Injection volume: 100 Detector: UV 270 for 4 min then UV 240 CHROMATOGRAM Retention time: 5

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Limit of detection: 20 jxg/g OTHER SUBSTANCES Simultaneous: saccharin, sorbic acid REFERENCE Puttemans,M.L.; Branders,C; Dryon,L.; Massart,D.L. Extraction of organic acids by ion-pair formation with tri-n-octylamine. Part 6. Determination of sorbic acid, benzoic acid, and saccharin in yogurt, J.Assoc.Off.Anal.Chem., 1985, 68, 80-82.

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Benzonatate
Molecular formula: C30H53NO11 Molecular weight: 603.75 CAS Registry No.: 104-31 -4 Merck Index: 1127 SAMPLE

Matrix: tissue Sample preparation: Homogenize 10 g tissue in 40 mL water, make alkaline with NaOH, extract twice with ether. Combine extracts and evaporate them to dryness, reconstitute the residue in 1 mL EtOH, inject a 50 |xL aliquot.
HPLC VARIABLES

Column: Reverse phase ODS Mobile phase: MeCNrIOO mM phosphoric acid 50:50 Column temperature: 40 Flow rate: 1 Injection volume: 50 Detector: UV 205
CHROMATOGRAM

Limit of detection: 200 ng/mL

KEYWORDS

liver; brain; blood; kidney

REFERENCE Cohan,J.A.; Manning,T.J.; Lukash,L.; Long,C; Ziminski,K.R.; Conradi,S.E. Two fatalities resulting from Tessalon (benzonatate), Vet.Hum.ToxicoL, 1986, 28, 543-544.

Benzoyl peroxide
Molecular formula: C14H10O4 Molecular weight: 242.23 CAS Registry No.: 94-36-0 Merck Index: 1149

SAMPLE

Matrix: blood Sample preparation: Condition a 100 mg Bond Elut C18 SPE cartridge with 2 mL MeCN and 2 mL 50 mM pH 7.5 phosphate buffer. Add 1 mL serum to the SPE cartridge, wash with 500 jxL 50 mM pH 7.5 phosphate buffer, elute with 1 mL MeCN, inject a 20 |xL aliquot of the eluate.
HPLC VARIABLES

Column: Capcell Pak C-18 SG-120 (Shiseido) Mobile phase: MeCN:water 50:50 Flow rate: 1.2 Injection volume: 20 Detector: UV 235
CHROMATOGRAM Retention time: 24 OTHER SUBSTANCES

Extracted: methyl methacrylate, N,N-dimethyl-p-toluidine

KEY WORDS

serum; dental material; methyl methacrylate polymer; plexiglass; horse; SPE

REFERENCE Shintani,H.; Tsuchiya,T.; Hata,Y; Nakamura,A. Solid phase extraction and HPLC analysis of toxic components eluted from methyl methacrylate dental materials, JAnaLToxicoL, 1993, 17, 73-78. SAMPLE

Matrix: food Sample preparation: Mix 1 g flour with 8.0 mL EtOH, shake vigorously for 1 min and extract by sonication for 15 min at room temperature. Centrifuge at 4000 rpm for 20 min, remove 4 mL supernatant, add 2 mL 100 mM KOH, mix in an ultrasonic water bath for 2 min at room temperature, dilute to 25 mL with water and set aside overnight. Filter (0.45 |xm) and inject a 50 |xL aliquot.
HPLC VARIABLES

Column: Dionex OmniPac PAX-100 anion exchange column Mobile phase: MeOH:3 mM sodium carbonate 2:98 Flow rate: 1.0 Injection volume: 50 Detector: UV 222
CHROMATOGRAM

Retention time: 2.5 Limit of detection: 19 ng/mL Limit of quantitation: 120 ng/mL

OTHER SUBSTANCES Interfering: benzoic acid

KEYWORDS

wheat flour; derivatization

REFERENCE Chen,Q.-C; Mou,S.-E; Hou,X.-R; Ni,Z.-M. Determination of benzoyl peroxide in wheat flour by ion chromatography with precolumn derivatization, J.Liq.Chromatogr.Rel.Technol., 1998, 21, 705-716. SAMPLE

Matrix: plastic Sample preparation: Leach with 20 mL water, MeOH, acetone, or THF at room temperature for 24 h, repeat. Combine solutions and add an equal volume of MeCN:water 50: 50, filter (0.45 \im) the supernatant, inject a 20 |xl aliquot.
HPLC VARIABLES

Column: 250 X 4.6 Capcell Pak SG-120 C18 (Shiseido) Mobile phase: MeCNrwater 48:52 Flow rate: 1 Injection volume: 20 Detector: UV 235 CHROMATOGRAM Retention time: 29.6
OTHER SUBSTANCES

Simultaneous: methyl methacrylate, N,N-dimethyl-p-toluidine

REFERENCE Shintani,H. HPLC analysis of toxic additives and residual monomer from dental plate, J.Liq.Chromatogr., 1995,18, 613-626.

Benzphetamine
Molecular formula: C17H21N Molecular weight: 239.36 CAS Registry No.: 156-08-1, 5411 -22-3 (HCI) Merck Index: 1151 LednicerNo.: 1 70

SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 250 X 5 Spherisorb S5W Mobile phase: MeOHrbuffer 90:10 (Buffer was 94 mL 35% ammonia and 21.5 mL 70% nitric acid in 884 mL water, adjust the pH to 10.1 with ammonia.)

Flow rate: 2 Injection volume: 20 Detector: UV 254

CHROMATOGRAM Retention time: 1.52 OTHER SUBSTANCES

Simultaneous: phendimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphentermine, norpseudoephedrine, phentermine, fenfluramine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, tyramine, trimethoxyamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephedrine, dimethylamphetamine, methamphetamine, mescaline, mephentermine, norpipanone, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamorphine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine Noninterfering: dopamine, levodopa, methyldopa, methyldopate, norepinephrine Interfering: pemoline, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine
REFERENCE Law,B-; Gill,R.; Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of forensic interest on a silica column using an aqueous methanol eluent, J.Chromatogr., 1984, SOl, 165-172. SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V CHROMATOGRAM Retention time: 1.9
OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, metnamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,I.; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax EX

Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, fiubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994, 18, 233-242.

Benzquinamide
Molecular formula: C22H32N2O5 Molecular weight: 404.51 CAS Registry No.: 63-12-7 Merck Index: 1154 Lednicer No.: 1 350

SAMPLE

Matrix: solutions Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 125 X 4.9 Spherisorb S5W silica Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in MeOH, pH 6.7

Flow rate: 2 Injection volume: 20

Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM Retention time: 1.2 OTHER SUBSTANCES

Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,

protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.

Benzthiazide
Molecular formula: C15H14CIN3O4S3 Molecular weight: 431.94 CAS Registry No.: 91-33-8 Merck Index: 1155

SAMPLE

Matrix: blood, feces, urine Sample preparation: Plasma. 3 mL Plasma + 500 jxL 1 fig/mL polythiazide in 10 mM NaOH + 1 mL 10 mM NaOH + 800 JJLL. 100 mM HCl + 10 mL dichloromethane, shake on a platform shaker for 20 min, centrifuge at -10 at 3000 rpm for 15 min, repeat extraction twice more. Combine all organic layers and evaporate them to dryness under a stream of nitrogen at 50, reconstitute the residue in 50 (JLL, vortex, inject whole amount. Urine. 5 mL Urine + 1 mL 2 jjig/mL polythiazide in 10 mM NaOH + 1 mL 10 mM NaOH + 2 mL 0.68% KH2PO4 adjusted to pH 6.1 + 10 mL dichloromethane, shake on a platform shaker for 20 min, centrifuge at -10 at 3000 rpm for 15 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 100 jxL, vortex, inject a 20 |JLL aliquot. Feces. Extract 20 g feces with 350 mL acetone, filter (Whatman No. 1 paper), evaporate to ca. 50 mL, make up to 100 mL with acetone. Remove a 10 mL aliquot and add it to 1 mL 300 jig/mL polythiazide in acetone, evaporate to dryness under nitrogen, dissolve in 10 mL MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES

Column: (xBondapak C18 Mobile phase: MeCN:glacial acetic acid:water 35:2:63 (plasma, feces) or MeCN:water 40: 60 (urine) Flow rate: 2 Injection volume: 10-50 Detector: UV 280
CHROMATOGRAM

Retention time: 7 (plasma), 5 (urine) Internal standard: polythiazide (9 (plasma), 8 (urine)) Limit of detection: 10 ng/mL (plasma) Limit of quantitation: 50 ng/mL (urine), 20 ng/mL (plasma)
KEYWORDS

plasma; pharmacokinetics
REFERENCE Meyer,M.C; Hwang,R; Straughn,A.B.; Rotenberg,K. HPLC determination of benzthiazide in biologic material, Biopharm.Drug Dispos., 1982, 3, 1-9. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: not specified Mobile phase: MeOH:water containing 200 mM ammonia and 200 mM glycolic acid 25:75 Flow rate: 0.8 Detector: MS, Hewlett-Packard 9000-300 quadrupole, thermospray, stem 108-115, tip 180205, ion source 276, filament on, negative ion mode
KEYWORDS

LC-MS

REFERENCE Kim,Y; Park,S.; Park,J.; Lee,W. Detection of benzthiazide by high-performance liquid chromatographythermospray mass spectrometry, J.Chromatogr.A, 1995, 689, 170-174. SAMPLE

Matrix: urine Sample preparation: 2 mL Urine + 0.5 g solid buffer I (pH 5-5.5), vortex 15 s, add 4 mL ethyl acetate, agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and vortex it with 2 mL 5% aqueous lead acetate for 10 s, centrifuge at 600 g for 5 min, remove and keep organic phase. 2 mL Urine + 0.5 g solid buffer II (pH 9-9.5), vortex 15 s, add 4 mL ethyl acetate, agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and combine it with previous organic layer. Evaporate to dryness at 50 under a stream of nitrogen, reconstitute in 300 |xL 50 [LgImL p-hydroxyethyltheophylline in MeOH, inject 5 |xL aliquot. (Solid buffer I was KH2PO4-Na2HPO4 99:1, solid buffer II was NaHCO3:K2CO3 3:2.)
HPLC VARIABLES

Column: 250 X 4.6 5 p,m HP Hypersil ODS (A) or HP LiChrosorb RP-18 (B) Mobile phase: Gradient. MeCN.buffer from 15:85 at 2 min to 80:20 at 20 min (Buffer was 50 mM NaH2PO4 containing 16 mM propylamine hydrochloride, adjusted to pH 3 with concentrated phosphoric acid.) Flow rate: 1 Injection volume: 5 Detector: UV 230, UV 275
CHROMATOGRAM

Retention time: 13.5 (A), 14.3 (B) Internal standard: p-hydroxyethyltheophylline (3.7 (A), 4.4 (B)) Limit of detection: 1000 ng/mL
OTHER SUBSTANCES

Extracted: furosemide, metolazone, amiloride, acetazolamide, chlorothiazide, hydrochlorothiazide, quinethazone, triamterene, hydroflumethiazide, chlorthalidone, dichlorphenamide, trichloromethiazide, methyclothiazide, cyclothiazide, polythiazide, bendroflumethiazide, ethacrynic acid, bumetanide, probenecid, spironolactone, canrenone, flumethiazide Noninterfering: acetaminophen, aspirin, caffeine, diflunisal, fenoprofen, ibuprofen, indomethacin, methocarbamol, naproxen, phenylbutazone, sulindac, tetracycline, theobromine, theophylline, tolmetin, trimethoprim, verapamil
REFERENCE Cooper,S.E; Masse,R.; Dugal,R. Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography, J.Chromatogr., 1989, 489, 65-88. SAMPLE

Matrix: urine Sample preparation: Make 5 mL urine alkaline (pH 9-10), add 2 g NaCl, extract twice with 6 mL ethyl acetate. Combine the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 200 jxL MeCN/water, inject a 10-20 jxL aliquot.
HPLC VARIABLES

Column: 100 X 4 5 |xm SGE 100 GL-4 C18P (Scientific Glass Engineering) Mobile phase: MeCN:MeOH:water:trifluoroacetic acid 4.5:10.5:85:0.5 Flow rate: 0.8 or 1 Injection volume: 10-20 Detector: MS, ZAB2-SEQ (VG), PSP source coupled to LC, source 250, probe 240-260, scan m/z 200-550 or UV 270

CHROMATOGRAM

Retention time: 7.2 Limit of detection: 50 ng (by MS)

OTHER SUBSTANCES

Extracted: amiloride, chlorthalidone, triamterene, furosemide, bendrofiumethiazide

REFERENCE Ventura,R; Fraisse,D.; Becchi,M.; Paisse,O.; Segura,J. Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography-mass spectrometry in doping control, J.Chromatogr., 1991, 562, 723-736. SAMPLE

Matrix: urine Sample preparation: Buffer urine to 4.9 by mixing with an equal volume of pH 4.9 200 mM sodium phosphate buffer. Inject a 40 |xL aliquot onto column A with mobile phase A, after 3 min backflush the contents of column A onto column B with mobile phase B and start the gradient. At the end of the run re-equilibrate for 10 min.
HPLCVARIABLES

Column: A 20 X 4 5 jxm Hypersil octadecylsilica ODS; B 200 X 4.6 5 ^m Shiseido SG-120 polymer-based C18 Mobile phase: A water; B Gradient. MeCN:buffer from 7:93 to 15:85 over 3.5 min, to 50: 50 over 8.5 min, maintain at 50:50 for 11 min (Buffer was 6.9 g NaH2PO4-H2O in 1 L water, pH adjusted to 3.1 with phosphoric acid.) Flow rate: 1 Injection volume: 40 Detector: UV 230
CHROMATOGRAM

Retention time: 17.6 Limit of detection: 500 ng/mL

OTHER SUBSTANCES

Extracted: acetazolamide, amiloride, bendrofiumethiazide, bumetanide, caffeine, carbamazepine, chlorothiazide, chlorthalidone, clopamide, dichlorfenamide, ethacrynic acid, furosemide, hydrochlorothiazide, metyrapone, probenecid, spironolactone, triamterene, trichlormethiazide
KEYWORDS

column-switching; optimum detection wavelengths vary for each drug

REFERENCE Saarinen,M.; Siren,H.; Riekkola,M.-L. A column switching technique for the screening of diuretics in urine by high performance liquid chromatography, J.Liq.Chromatogr., 1993, 16, 4063-4078. SAMPLE

Matrix: urine Sample preparation: 5 mL Urine + 50 |xL 100 |xg/mL 7-propyltheophylline in MeOH + 200 U.L ammonium chloride buffer + 2 g NaCl, extract with 6 mL ethyl acetate by rocking at 40 movements/min for 20 min and centrifuging at 800 g for 5 min, repeat extraction, combine organic layers, evaporate to dryness at 40 under a stream of nitrogen. Reconstitute in 200 |xL MeCN:water 15:85 and inject 20 (xL aliquots. (Ammonium chloride buffer was 28 g ammonium chloride in 100 mL water with the pH adjusted to 9.5 with concentrated ammonia solution.)
HPLC VARIABLES

Column: 75 X 4.6 3 jxm Ultrasphere ODS

Mobile phase: Gradient. MeCN: 100 mM ammonium acetate adjusted to pH 3 with concentrated phosphoric acid. From 10:90 to 15:85 over 2 min to 55:45 over 3 min to 60:40 over 3 min. Kept at 60:40 for 1 min, decreased to 10:90 over 1 min and equilibrated at 10:90 for 2 min. Flow rate: 1 Injection volume: 20 Detector: UV 270
CHROMATOGRAM

Retention time: 6.5 Internal standard: 7-propyltheophylline (4.5) Limit of detection: 100 ng/mL
OTHER SUBSTANCES

Simultaneous: xipamide, bumetanide, acetazolamide, amiloride, bendroflumethiazide, buthiazide, caffeine, canrenone, chlorthalidone, clopamide, cyclothiazide, diclofenamide, ethacrynic acid, furosemide, hydrochlorothiazide, mesocarb, morazone, piretanide, polythiazide, probenecid, spironolactone, torsemide, triamterene
REFERENCE Ventura,R.; Nadal,T.; Alcalde,R; Pascual,J.A.; Segura,J. Fast screening method for diuretics, probenecid and other compounds of doping interest, J.Chromatogr.A, 1993, 655, 233-242.

Benztropine mesylate
Molecular formula: C22H29NO4S Molecular weight: 403.54 CAS Registry No.: 132-17-2, 86-13-5 (free base) Merck Index: 1156

SAMPLE

Matrix: blood Sample preparation: 10 mL Plasma or whole blood + 1 mL 1 M NaOH, extract twice with 10 mL hexane for 30 min. Remove the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 1 mL 100 mM HCl, add 5 mL chloroform, vortex for 1 min, centrifuge. Remove a 4.5 mL aliquot of the organic layer and evaporate it to dryness, reconstitute the residue in 100 |xL mobile phase, inject a 50 IxL aliquot. (It is implied, but not explicitly stated in the paper, that this extraction procedure works for this compound.)
HPLC VARIABLES

Column: 10 fim Micropak CN (Varian) Mobile phase: MeCN:20 mM ammonium acetate 90:10 Flow rate: 2.5 Injection volume: 50 Detector: UV 254
CHROMATOGRAM

Retention time: 13.2 Limit of detection: 10 ng/mL

OTHER SUBSTANCES
x

Simultaneous: acetophenazine, amitriptyline, butaperazine, carphenazine, chlorpromazine, fluphenazine, haloperidol, imipramine, mesoridazine, nortriptyline, orphenadrine, piperacetazine, promazine, promethazine, thioridazine, thiothixene, trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine
KEY WORDS

plasma; whole blood

REFERENCE Curry,S.H.; Brown,E.A.; Hu,O.Y.-R; Perrin,J.H. Liquid chromatographic assay of phenothiazine, thioxanthene and butyrophenone neuroleptics and antihistamines in blood and plasma with conventional and radial compression columns and UV and electrochemical detection, J.Chromatogr., 1982, 231, 361-376. SAMPLE

Matrix: blood Sample preparation: 2 mL Plasma + 150 jxL 200 ng/mL desipramine hydrochloride + 150 IxL 5 M NaOH, vortex, add 1 mL ethylene glycol, vortex, add 10 mL hexane, shake on a rotary shaker at 30 rpm for 30 min, centrifuge at 1000 g at 4. Remove the organic layer and add it to 300 |xL 100 mM HCl, shake at high speed for 20 min, centrifuge, inject a 200 |xL aliquot of the aqueous layer.
HPLC VARIABLES

Column: 150 X 3.9 5 |xm Spherisorb C8

Mobile phase: MeCN:buffer 60:40 (Buffer was 1.5 mL triethylamine in 1 L water adjusted to pH 3.0 with 85% phosphoric acid.) Flow rate: 1.5 Injection volume: 200 Detector: UV 199
CHROMATOGRAM

Retention time: 7.0 Internal standard: desipramine hydrochloride (5.3) Limit of quantitation: 0.25 ng/mL
OTHERSUBSTANCES

Simultaneous: hyoscyamine, orphenadrine, bromocriptine, biperiden Noninterfering: amantadine, carbidopa, levodopa

KEYWORDS

plasma
REFERENCE Selinger,K.; Lebel,G.; Hill,H.M.; Discenza,C. High-performance liquid chromatographic method for the analysis of benztropine in human plasma, J.Chromatogr., 1989, 491, 248-252. SAMPLE

Matrix: blood, tissue Sample preparation: Blood or serum. 1 mL Blood or serum + 1 jxg cianopramine + 1 mL water, vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexane:l-butanol 95:5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it to 100 JULL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic layer and inject a 30 JULL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver homogenate + 10 jxg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it to 400 |xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic layer and inject a 30 |xL aliquot of the aqueous layer.
HPLC VARIABLES

Guard column: 15 X 3.2 7 |xm RP-18 Newguard (Applied Biosystems) Column: 100 X 4.6 5 \xm Brownlee Spheri-5 RP-18 Mobile phase: MeCN:100 mM NaH2PO4:diethylamine 40:57.5:2.5 Flow rate: 2 Injection volume: 30 Detector: UV 220
CHROMATOGRAM

Retention time: 22.75 Internal standard: cianopramine (8.93)

OTHER SUBSTANCES

Simultaneous: amoxapine, brompheniramine, chlorpheniramine, chlorpromazine, clomipramine, cyproheptadine, desipramine, diphenhydramine, dothiepin, doxepin, fluoxetine, haloperidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine, methadone, metoclopramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, northiaden, nortriptyline, pentobarbital, pheniramine, propoxyphene, propranolol, protriptyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazodone, trihexiphenidyl, triprolidine Noninterfering: dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine, trifluoperazine Interfering: promethazine, trimipramine, amitriptyline

KEY WORDS

serum; whole blood; liver

REFERENCE McIntyreJ.M.; King,C.V.; Skafidis,S.; Drummer,O.H. Dual ultraviolet wavelength high-performance liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and some selected metabolites, J.Chromatogr., 1993, 621, 215-223. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject
a 10 JULL aliquot. HPLC VARIABLES

Column: 300 X 3.9 10 jim u-Bondapak C18 Mobile phase: MeOHracetic acid:triethylamine:water 60:1.5:0.5:38 Flow rate: 1.5 Injection volume: 10 Detector: UV CHROMATOGRAM Retention time: k' 2.10
REFERENCE Roos,RW.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenpro-

porex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE HiIl5D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994, 18, 233-242.

Benzydamine
Molecular formula: C19H23N3O Molecular weight: 309.41 CAS Registry No.: 642-72-8, 132-69-4 (HCI) Merck Index: 1157 Lednicer No.: 1 323 SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 jxm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 215.8 CHROMATOGRAM Retention time: 14.955 KEYWORDS whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Benzyl alcohol
Molecular formula: C7H8O Molecular weight: 108.14 CAS Registry No.: 100-51 -6 Merck Index: 1159

SAMPLE

Matrix: bulk Sample preparation: Dissolve 600 fig 7-[(imidazolemethanoyl)methoxy]-4-methylcoumarin in 2.5 mL toluene and 750 JJLL MeCN, add 20 |xL benzyl alcohol, add 150 |xL 93.3 jxg/mL 4-dimethylaminopyridine in MeCN, mix vigorously, heat at 60 for 1.5 h, cool, evaporate to dryness under a stream of nitrogen at room temperature, reconstitute the residue in 300 |xL mobile phase, inject a 5 IULL aliquot. (Preparation of 7-[(imidazolemethanoyl)methoxy]-4-methylcoumarin is as follows. Stir 102.3 mg 7-(carboxymethoxy)4-methylcoumarin in 7 mL THF, add 70.9 mg l,l'-carbonyldiimidazole in one portion, reflux for 30 min, stir at room temperature for 5 h. Filter and dry the solid under reduced pressure to obtain 7-[(imidazolemethanoyl)methoxy]-4-methylcoumarin as a white solid (mp 161-162). Fluorescence detection can also be used.)
HPLC VARIABLES

Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: MeCN:MeOH:100 mM pH 5.5 ammonium acetate buffer 50:1.5:48.5

Flow rate: 0.7 Injection volume: 5

Detector: MS, Hewlett-Packard 5989A, thermospray interface, filament-assisted ionization mode, ion source 280, probe stem 112, probe tip 235-245 CHROMATOGRAM Retention time: 7 Limit of detection: 0.8 ng
KEYWORDS

derivatization
REFERENCE Phillips,L.R.; Supko,J.G.; Wolfe,T.L.; Malspeis,L. Precolumn derivatization of hydroxy compounds with 7-[(imidazolemethanoyl)methoxy]-4-methylcoumarin (IMMC) and LC/TSP-MS of the resulting esters, ProcAm.Soc.Mass Spectrom., 1995, 43, 163-164.

Benzyl benzoate
Molecular formula: C14H12O2 Molecular weight: 212.25 CAS Registry No.: 120-51 -4 Merck Index: 1162

SAMPLE

Matrix: formulations Sample preparation: Dilute 0.5 mL of nanocapsules suspension 1:200 with MeCN, filter, inject an aliquot. Alternatively, evaporate 5 mL of a nanocapsule suspension to dryness and dissolve the residue in 150 mL dichloromethane or ethyl acetate, dry over anhydrous sodium sulfate. Evaporate to dryness under reduced pressure, take up the residue in 50 mL MeOH, dilute 1:20 with MeOH, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4 5 \im Nucleosil C18 Mobile phase: MeCN: water 75:25 Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 4.71 OTHER SUBSTANCES Simultaneous: progesterone Noninterfering: poly-e-caprolactone
KEYWORDS

nanocapsules
REFERENCE Benali,S.; Tharasse-Bloch,C; Andre; Verite,R; Duclos,R.; Lafont,O. Determination od progesterone in nanocapsules by high performance liquid chromatography, J.Liq.Chromatogr.Rel.Technol., 1997,20, 3233-3243. SAMPLE

Matrix: formulations Sample preparation: Injections. Extract 2 mL with EtOHrwater 85:15, make up extracts to 100 mL with EtOH:water 85:15, remove a 2 mL aliquot and add it to 1 mL 1 mg/mL hydrocortisone in EtOH. Dilute this mixture to 50 mL with EtOH:water 50:50, inject an aliquot. Suspensions. Dilute 2 mL suspension to 100 mL with EtOH, filter (if necessary), remove a 2 mL aliquot and add it to 1 mL 1 mg/mL hydrocortisone in EtOH. Dilute this mixture to 50 mL with EtOH, inject an aliquot.
HPLCVARIABLES

Column: 300 X 4 ixBondapak CN Mobile phase: MeOH:20 mM KH2PO4 30:70 Flow rate: 2 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 6.5

Internal standard: hydrocortisone (2.5)

OTHER SUBSTANCES

Simultaneous: medroxyprogesterone acetate Noninterfering: polyethylene glycol 4000, mystristyl-gamma-picolinium chloride, methylcellulose, thimerosal Interfering: progesterone
REFERENCE Das Gupta,V. Quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone by reversed-phase high-pressure liquid chromatography, J.Pharm.Sci., 1982, 71, 294-297. SAMPLE

Matrix: formulations Sample preparation: Oils. 1 mL Sample + 25 mL MeOHrwater 90:10, shake vigorously for 5 min, centrifuge, inject a 10 |xL aliquot of the supernatant. Tablets. Grind a tablet to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first 5 mL of nitrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous). Make up 5 mL to 50 mL with MeOH, filter (0.45 jxm), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Zorbax ODS Mobile phase: MeOH:water 75:25 Flow rate: 1.5 Injection volume: 10 Detector: UV 240 CHROMATOGRAM Retention time: 11.8 Limit of detection: 5 fxg/mL
OTHER SUBSTANCES

Simultaneous: aspirin, caffeine, formebolone, benzyl alcohol, testolactone, cortisone, fluoxymesterone, norethindrone, oxandrolone (UV 210), boldenone, ethisterone, methandrostenolone, nandrolone, norgestrel, testosterone, dehydroepiandrosterone (UV 210), mibolerone, methyltestosterone, methandriol (UV 210), norethindrone acetate, calusterone, mesterolone (UV 210), norethandrolone, trenbolone acetate, nandrolone acetate, testosterone acetate, stanozolol, oxymetholone, nandrolone propionate, methenolone acetate, testosterone propionate
KEYWORDS

oils; tablets; suspensions

REFERENCE Walters,M.J.; Ayers,R.J.; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry, JAssoc.OffAnal.Chem., 1990, 73, 904-926. SAMPLE

Matrix: formulations Sample preparation: Weigh out 50 mg formulation, add 5 mL 1.5 mg/mL benzophenone in MeOH, make up to 50 mL with MeOH. Dilute 1 mL of this solution to 10 mL with MeOH, filter (0.45 |xm PTFE membrane), inject a 10 fxL aliquot.
HPLC VARIABLES

Guard column: 30 mm long Brownlee guard column

Column: 220 X 4.6 5 |mm C18 (Brownlee) Mobile phase: MeCN:water 60:40 Flow rate: 2 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: 4.5 Internal standard: benzophenone (3.3) Limit of quantitation: 7000 ng/mL OTHER SUBSTANCES Simultaneous: benzocaine
KEYWORDS

dermatological preparations
REFERENCE Gigante,B.; Barros,A.M.V.; Teixeira,A.; Marcelo-Curto,M.J. Separation and simultaneous high-performance liquid chromatographic determination of benzocaine and benzyl benzoate in a pharmaceutical preparation, J.Chromatogr., 1991, 549, 217-220. SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH at a concentration of 100 |xg/mL, inject a 5 |JLL aliquot.
HPLC VARIABLES

Guard column: 70 X 2.1 CO:Pell ODS Column: 300 X 3.9 Bondex C18 (Phenomenex) Mobile phase: MeOH:water 85:15 Flow rate: 1 Injection volume: 5 Detector: UV 254 CHROMATOGRAM Retention time: 5
OTHER SUBSTANCES

Also analyzed: boldenone, testosterone, nandrolone, and their esters

REFERENCE Noggle,ET.,Jr.; Clark,C.R.; DeRuiter,J. Liquid chromatographic and mass spectral analysis of the anabolic 17-hydroxy steroid esters, J.Chromatogr.ScL, 1990, 28, 263-268. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 3.9 5 jim Zorbax BP-ODS Mobile phase: MeCN:50 mM pH 7.2 sodium phosphate buffer 40:60 Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 8

Internal standard: benzyl benzoate OTHER SUBSTANCES Extracted: HO-221 KEYWORDS benzyl benzoate is IS REFERENCE Kondo,N.; Iwao,T.; Hirai,K.-L; Fukuda,M.; Yamanouchi,K; Yokoyama,K; Miyaji,M.; Ishihara,Y.; Kon,K; Ogawa,Y; Mayumi,T. Improved oral absorption of enteric coprecipitates of a poorly soluble drug, J.Pharm.ScL, 1994, 83, 566-570.

Bepridil
Molecular formula: C24H34N2O Molecular weight: 366.55 CAS Registry No.: 64706-54-3, 74764-40-2 (HCI monohydrate) Merck Index: 1188 LednicerNo.: 3 46

SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanohn-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |ULL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 fim NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)

Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 249

CHROMATOGRAM

Retention time: 18.30 Limit of detection: <120 ng/mL

KEYWORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol;

aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,P.; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 x 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 202.8 CHROMATOGRAM Retention time: 19.503
KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: formulations Sample preparation: Weigh out ground tablets or capsules equivalent to 150 mg bepridil.HCl, add 150 mL MeCN, shake for 30 min, dilute to 200 mL with MeCN, filter (Schleicher & Schiill paper, grade 588), inject a 20 jxL aliquot.

HPLC VARIABLES

Column: 300 X 4.6 10 |xm jjuBondapak C18 Mobile phase: MeCNibuffer 580:405 (Buffer was 1.1 g sodium 1-heptanesulfonate in 405 mL water, adjust to pH 2.37 with glacial acetic acid (ca. 15 mL).) Column temperature: 35 Flow rate: 1.3 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 6.8
OTHER SUBSTANCES

Simultaneous: impurities, benzaldehyde, N-benzylaniline, benzoic acid

KEY WORDS

stability-indicating; rugged; capsules; tablets

REFERENCE Renzi,N.L.; Fronheiser,M.E.; Duong,H.T.; Fulton,D.J.; Rabinowitz,M. Stability-indicating high-performance liquid chromatography assay for bepridil hydrochloride drug substance and drug products, J.Chromatogn, 1989, 462, 398-405. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Chirex 3014 (Phenomenex) Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 62:35:3 (EtOH/trifluoroacetic acid was premixed 20:1.) Flow rate: 0.7-1 Injection volume: 20 Detector: UV 250
KEY WORDS

chiral; a = 1.22 for enantiomers

REFERENCE Cleveland,*!. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical racemates, J.Liq.Chromatogr., 1995, 18, 649-671. SAMPLE

Matrix: solutions Sample preparation: Prepare a 100 |xM solution in buffer, inject a 20 JJLL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 column containing riboflavin binding proteins (Prepare as follows. Add riboflavin to saturate protein of egg yolk, homogenize with 3 volumes buffer, centrifuge, add the supernatant to a 500 X 30 column of DEAE-cellulose (Whatman) equilibrated with buffer, wash extensively with buffer to remove bound protein, elute riboflavin binding proteins (RFBP) with buffer containing 200 mM NaCl (RFBP has intense yellow color, absorption at 455 nm). Purify RFBP on a Sephadex G-100 column with 50 mM pH 7.5 Tris-HCl buffer as eluent, remove the bound riboflavin by extensive dialysis at pH 3.0. Add 4.5 g N,N-disuccinylimidyl carbonate to 3 g Nucleosil 5NH2 slurried in MeCN, filter, wash with MeCN, wash with 50 mM pH 7.5 phosphate buffer. Suspend 300 mg RFBP in 50 mM phosphate buffer, add the activated silica, mix gently for 2 h using a rotary evaporator, filter, wash with sterile water, wash with isopropanol:water 1:2, pack in a 100 X 4.6 column.) (Buffer was 100 mM pH 5.3 sodium acetate.)

Mobile phase: EtOH:50 mM pH 5.5 KH2PO4 5:95 Flow rate: 0.8 Injection volume: 20 Detector: UV CHROMATOGRAM Retention time: k' 11.96 OTHER SUBSTANCES Simultaneous: lorazepam, manidipine, nicardipine, oxazepam KEYWORDS chiral; a = 1.21 REFERENCE Massolini,G.; De Lorenzi,E.; Ponci,M.C; Gandini,C; Caccialanza,G.; Monaco,H-L- Egg yolk riboflavin binding protein as a new chiral stationary phase in high-performance liquid chromatography, J.ChromatogrA, 1995, 704, 55-65.

Berberine
Molecular formula: C20H18NO4 Molecular weight: 336.37 CAS Registry No.: 2086-83-1 Merck Index: 1192

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210
OTHER SUBSTANCES

Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, py-

rilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994, 18, 233-242.

Besipirdine
Molecular formula: C16H17N3 Molecular weight: 251.33 CAS Registry No.: 119257-34-0, 130953-69-4 (HCI), 119257-40-8 (maleate) Merck Index: 1223

SAMPLE

Matrix: cell cultures Sample preparation: Mix 1 mL cell culture with 50 J U L L 1 mg/mL IS in MeOH and 1 mL saturated aqueous sodium bicarbonate, add 3 mL ethyl acetatexyclohexane 50:50, invert at 18 cycles/min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 2 mL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 2jxm Hypersil-Phenyl Mobile phase: MeCN:buffer 85:15 (Buffer was 5.8 mM triethylammonium formate adjusted to pH 2.5 with 90% formic acid.) Flow rate: 1 Injection volume: 20 Detector: UV 270
CHROMATOGRAM

Retention time: 10 Internal standard: 3-ethyl-N-methyl-N-(4-pyridinyl)-lH-indol-l-amine hydrochloride

OTHER SUBSTANCES Extracted: metabolites KEYWORDS

details of semipreparative HPLC

REFERENCE Rao,G.R; Davis,P.J. Microbial models of mammalian metabolism. Biotransformations of HP 749 (besperidine) using Cunninghamella elegans, Drug Metab.Dispos., 1997, 25, 709-715.

Beta-carotene
Molecular formula: C20H56 Molecular weight: 536.88 CAS Registry No.: 7235-40-7 Merck Index: 1902

SAMPLE

Matrix: blood Sample preparation: Centrifuge 200 |xL serum, add 200 |xL IS and 200 |xL EtOH, mix on orbital shaker for 5 min, add 200 |xL water and 500 |JLL hexane, mix for 10 min, centrifuge at 2000 g for 10 min at 17, remove 300 JLJLL upper organic layer. Re-extract with 300 jxL hexane, mix for 10 min, centrifuge at 4000 g for 10 min at 17, remove 300 |xL upper organic layer. Combine the organic layers, and evaporate them to dryness under vacuum in 15 min. Reconstitute the residue with 300 JLJLL MeOH:EtOH:hexane 88:10:2, vortex for 10 min, inject a 40 u,L aliquot.
HPLC VARIABLES

Column: 100 X 4.6 3 |xm Adsorbosphere HS C18 + 150 X 4.6 3 fxm Adsorbosphere HS C18 in series Mobile phase: Gradient. A was MeCN:MeOH 60:40 containing 0.05% acetic acid. B was MeCN:MeCN:dichloromethane 45.6:30.4:24 containing 0.04% acetic acid. A:B 100:0 for 7 min then 0:100 for 10.4 min (step gradient), re-equilibrate at initial conditions for 5.6 min. Column temperature: 37 Flow rate: 0.9 Injection volume: 40 Detector: UV 450
CHROMATOGRAM

Retention time: 16.5 Internal standard: tocol (UV 292) (10.1), echinenone (UV 450) (12.8) Limit of detection: 10 ng/mL
OTHER SUBSTANCES

Extracted: canthaxanthine (UV 473), a-carotene (UV 450), p-cryptoxanthine (UV 450), Iutein (UV 450), lycopene (UV 473), vitamin A (UV 325), vitamin E (UV 292), zeaxanthin (UV 450), nonidentified carotenoids
KEYWORDS

serum
REFERENCE Steghens,J.-R; van Kappel,A.L.; Riboli,E.; Collombel,C. Simultaneous measurement of seven carotenoids, retinol and a-tocopherol in serum by high-performance liquid chromatography, J.Chromatogr.B, 1997, 694, 71-81. SAMPLE

Matrix: blood Sample preparation: Add 1 mL 1 |xg/mL retinyl palmitate, 1 jjug/mL retinyl palmitate, and 25 jmg/mL a-tocopheryl acetate in EtOH to 1 mL serum or plasma while continuously vortexing, add 3 mL hexane, vortex for 2 min, centrifuge at 2500 g for 2 min, remove the upper phase, add 2 mL hexane to the lower layer, repeat extraction. Combine the upper layers and evaporate them to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 uL mobile phase, inject a 40 JJIL aliquot.

HPLC VARIABLES

Guard column: C18 (Waters) Column: 5 |xm Biophase ODS C18 (Bioanalytical Systems) Mobile phase: MeCN:chloroform:isopropanol:water 78:16:3.5:2.5 Flow rate: 2 Injection volume: 40 Detector: UV 460 (UV 292 for tocopherol)
CHROMATOGRAM

Retention time: 16.46 Internal standard: retinyl acetate (3.07), retinyl palmitate (18.66), a-tocopheryl acetate (8.33)
OTHER SUBSTANCES

Extracted: vitamin A (retinol), vitamin E (a-tocopherol), gamma-tocopherol, a-carotene, Iycopene, cryptoxanthin

KEYWORDS

serum; plasma
REFERENCE Kaplan,L.A.; Miller,JA.; Stein,EA.; Stampfer,M.J. Simultaneous, high-performance liquid chromatographic analysis of retinol, tocopherols, lycopene, and a- and (3-carotene in serum and plasma, Methods EnzymoL, 1990, 189, 155-167. SAMPLE

Matrix: blood Sample preparation: 250 |xL Serum + 25 |JLL 80 |xg/mL tocol in EtOH + 250 JJLL 20 |xg/mL BHT (butylated hydroxytoluene) in EtOH +1.5 mL hexane, vortex for 1 min, remove 1 mL of upper layer, add 500 jxL hexane, vortex for 1 min, remove 300 |xL of upper layer. Combine the hexane extracts, evaporate to dryness under a stream of inert gas. Reconstitute in 250 |ULL 20 (xg/mL BHT in EtOH, sonicate, centrifuge if necessary, inject a 25 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 [xm Vydac 201TP54 (wide pore, polymerically bonded C18) Mobile phase: Gradient. A was MeOH:n-butanol:water 75:10:15 containing 50 mM ammonium acetate, pH 5.5. B was MeOH:n-butanol:water 88:10:2 containing 50 mM ammonium acetate, pH 5.5. A:B 100:0 for 3 min, to 0:100 over 15 min, maintain at 0:100 for 17 min Injection volume: 25 Detector: UV 325 for 7 min, UV 295 for 13 min, UV 450 for 14 min or E, glassy carbon electrode, Ag/AgCl reference electrode +1050 mV for retinol, +900 mV for tocol, +750 mV for a-tocopherol, +700 mV for (3-carotene
CHROMATOGRAM

Retention time: 31 Internal standard: tocol (13) Limit of detection: 2.1 |xg/mL (E), 29 |xg/mL (UV)
OTHER SUBSTANCES

Extracted: vitamin A (retinol), vitamin E (a-tocopherol), gamma-tocopherol, lutein, zeaxanthin, cryptoxanthin, a-carotene, 9-cis-p-carotene
KEYWORDS

serum

REFERENCE MacCrehan,W.A. Determination of retinol, ct-tocopherol, and (3-carotene in serum by liquid chromatography, Methods EnzymoL, 1990, 189, 172-181. SAMPLE

Matrix: blood Sample preparation: 200 |xL Serum + 100 (xL EtOH + 100 |xL a-tocopheryl acetate in EtOH, vortex for 5 s, add 500 |xL hexane, vortex for 2 min, centrifuge at 700 g for 5 min. Remove 250 |xL of the hexane layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200 |xL mobile phase, mix for 2 min, inject a 50 |xL aliquot.
HPLC VARIABLES

Guard column: 30 X 4.6 10 jjim Spheri-10 RP18 Column: 150 X 4.6 5 |xm Ultrasphere ODS Mobile phase: MeCN:dichloromethane:MeOH 70:20:10 Flow rate: 1.2 Injection volume: 50 Detector: UV 325 for 3.5 min, UV 291 for 4.5 min, UV 450 for 6 min
CHROMATOGRAM

Retention time: 11.79 Internal standard: a-tocopheryl acetate (6.30) Limit of detection: 50 nM OTHER SUBSTANCES Extracted: vitamin A, vitamin E
KEY WORDS

protect from light; serum

REFERENCE Arnaud,J.; Fortis,!; Blachier,S.; Kia,D.; Favier,A. Simultaneous determination of retinol, a-tocopherol and |3-carotene in serum by isocratic high-performance liquid chromatography, J.Chromatogr., 1991, 572, 103-116. SAMPLE

Matrix: blood Sample preparation: 2.5 mL Plasma + 2.5 mL 18 ng/mL ISl and 10 ng/mL IS2 in EtOH, shake vigorously for 20 s, centrifuge at 1200 g for 5 min, add 5 mL diethyl ether, shake vigorously, centrifuge for 5 min, extract twice more with 5 mL ether. Combine ether layers, wash with 15 mL 5% NaCl, dry over sodium sulfate, evaporate to dryness under vacuum at 35. Dissolve residue in 1-2 mL dichloromethane, filter (0.45 |xm). Evaporate to dryness under a stream of nitrogen, make up to 100 JLJLL with MeCN:MeOH:dichloromethane:hexane 45:10:22.5:22.5, inject a 20 (xL aliquot.
HPLC VARIABLES

Guard column: 30 X 4.6 5 ^m Spheri-5-C18 (Brownlee) Column: 250 X 4.6 5 |xm Microsorb C18 (Rainin) Mobile phase: Gradient. MeCN:MeOH:dichloromethane:hexane 85:10:2.5:2.5 for 10 min then to 45:10:22.5:22.5 over 30 min, re-equilibrate for 15 min Flow rate: 0.7 Injection volume: 20 Detector: UV 470 CHROMATOGRAM Retention time: 34

Internal standard: ISl ethyl p-apo-8'-carotenate (18), IS2 (3R)-8'-apo-p-carotene-3,8'-diol (5)

OTHERSUBSTANCES

Extracted: carotenoids, vitamin A (retinol), vitamin E (a-tocopherol)

KEYWORDS

plasma; handle under yellow lights

REFERENCE Khachik,R; Beecher,G.R.; Goli,M.B.; Lusby,W.R.; Smith,J.C.,Jr. Separation and identification of carotenoids and their oxidation products in the extracts of human plasma, Anal.Chem., 1992, 64, 21112122. SAMPLE

Matrix: blood Sample preparation: 200 JULL Serum or plasma + 200 jxL 25 |xg/mL tocopheryl acetate in EtOH, vortex, add 400 |JLL butanol:ethyl acetate 50:50, mix for 1 min, add 20 mg sodium sulfate, vortex for 1 min, let stand at -20 for 20 min, centrifuge at 15000 g for 2 min, inject a 10 JJLL aliquot of the upper organic layer.
HPLC VARIABLES

Guard column: 5 jim C18 Column: 110 X 4.7 5 jxm Partisphere 5 C18 (Whatman) Mobile phase: MeOH.butanol:water 89.5:5:5.5 Column temperature: 45 Flow rate: 1.5 Injection volume: 10 Detector: UV 340 for 3 min, UV 290 for 1.5 min, UV 280 for 10.5 min, UV 450 for 7 min
CHROMATOGRAM

Retention time: 20.1 Internal standard: tocopheryl acetate (5.3) Limit of detection: 100 ng/mL
OTHER SUBSTANCES

Extracted: a-carotene, lycopene, 8-tocopherol, gamma-tocopherol, vitamin A, vitamin E, xanthophyll

KEY WORDS

serum; plasma; protect from light

REFERENCE Lee,B.L.; Chua,S.C; Ong,H.Y; Ong,C.N. High-performance liquid chromatographic method for routine determination of vitamins A and E and (3-carotene in plasma, J.Chromatogr., 1992, 581, 41-47. SAMPLE

Matrix: blood Sample preparation: Dilute 1 mL serum 0.5-5 times with saline. Add 1 mL EtOH to 1 mL diluted serum drop wise while vortexing, add 1.5 mL n-heptane, vortex for 1 min, centrifuge at 3000 rpm (Labofuge) for 15 min. Remove 1.3 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 40 IULL MeCN:THF 50:50, inject a 5 ^L aliquot.
HPLC VARIABLES

Column: 200 X 2.1 5 |xm ODS Hypersil Mobile phase: MeCN:water:THF 81.3:5.7:13

Column temperature: 40 Flow rate: 0.4 Injection volume: 5 Detector: UV 450 CHROMATOGRAM Retention time: 14.230 Limit of detection: 1 ng
OTHER SUBSTANCES

Extracted: Vitamin A (retinol), Vitamin E (a-tocopherol), probucol, gamma-tocopherol, Iycopene, a-carotene, metabolites
KEYWORDS

serum
REFERENCE Schafer Elinder,L.; Walldius,G. Simultaneous measurement of serum probucol and lipid-soluble antioxidants, J.LipidRes., 1992, 33, 131-137. SAMPLE

Matrix: blood Sample preparation: 20-500 |xL Serum + 2 volumes EtOH + 1 mL ethyl acetate + 4-7 |xL of a solution containing 16 mg/mL tocopheryl acetate, 2-3 |xg/mL canthaxanthin, and 10 (xg/mL retinoic acid, vortex for 30 s, centrifuge for 30 s, extract the pellet twice with 0.51 mL portions of ethyl acetate, extract the pellet with 0.5-1 mL hexane. Combine the supernatants, add 500 |JLL water, vortex, centrifuge. Remove the upper organic layer and evaporate it to dryness under a stream of argon, reconstitute the residue in 100 |LJLL MeOH:dichloromethane 2:1, inject a 10-90 |xL aliquot,
HPLC VARIABLES

Guard column: C18 (Upchurch) Column: 300 X 3.9 5 |xm Resolve C18 (Waters) Mobile phase: MeCN:dichloromethane:MeOH:l-octanol 90:15:10:0.1 Flow rate: 1 Injection volume: 10-90 Detector: UV 450
CHROMATOGRAM

Retention time: 21 Internal standard: tocopheryl acetate, canthaxanthin, retinoic acid

OTHER SUBSTANCES

Extracted: carotenoids, vitamin A (UV 325), vitamin E (UV 290)

KEY WORDS

protect from light; serum

REFERENCE Barua,A.B.; Kostic,D.; Olson,J.A. New simplified procedures for the extraction and simultaneous highperformance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum, J.Chromatogr., 1993, 617, 257-264. SAMPLE Matrix: blood

Sample preparation: 500 jxL Serum or plasma + 500 uX EtOH containing 4.27 fxM retinyl acetate and 0.31 |xM echinenone, rotamix for 30 s, add 2 mL n-hexane, rotamix for 30 s,

centrifuge at 2000 g for 2 min, repeat extraction with 2 mL n-hexane. Combine the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 50 |xL THF, make up to 200 ^L with EtOH, inject a 50 [xL aliquot.
HPLC VARIABLES

Guard column: 50 X 4.6 5 |xm Spherisorb ODSl Column: 250 X 4.6 5 jxm Spherisorb ODSl Mobile phase: Gradient. A was MeCNrMeOH 20:80 containing 100 mM ammonium acetate. B was 100 mM ammonium acetate in water. A:B from 90:10 to 100:0 over 12 min, maintain at 100:0 for 10 min, re-equilibrate at initial conditions for 5 min Flow rate: 2 Injection volume: 50 Detector: UV 325 for 7.5 min, UV 292 for 5.5 min, then UV 450
CHROMATOGRAM

Retention time: 19.50 Internal standard: retinyl acetate (5.96), echinenone (15.15) Limit of detection: 0.13 \xM
OTHER SUBSTANCES

Extracted: cryptoxanthin, lutein, lycopene, vitamin A, vitamin E

KEYWORDS

plasma; protect from light; serum

REFERENCE Zaman,Z.; Fielden,R; Frost,P.G. Simultaneous determination of vitamins A and E and carotenoids in plasma by reversed-phase HPLC in elderly and younger subjects, Clin.Chem., 1993, 39, 2229-2234. SAMPLE

Matrix: blood Sample preparation: 200 jxL Plasma or serum + 200 jxL 850 ng/mL retinyl acetate in EtOH, mix for 1 min, add 1 mL 0.4 g/L BHT (2,6-di-tert-butyl-4-methylphenol) in nhexane, shake on a mechanical shaker for 10 min, centrifuge at 2000 g for 5 min, remove 800 (xL of the supernatant, evaporate to dryness at 40 under a stream of nitrogen, reconstitute in 100 uL MeCN:THF:MeOH 68:22:7, inject a 15 fiL aliquot.
HPLC VARIABLES

Guard column: 15 X 3.2 7 |xm Lichrosorb RP18 Column: 250 X 4.6 5 |xm Nucleosil 100-5 C18 Mobile phase: MeCN:THF:MeOH 68:22:7 made up to 100 with 1% ammonium acetate Flow rate: 1.5 Injection volume: 15 Detector: UV 325 for 3 min, UV 450 for 1.9 min, UV 290 for 2.5 min, UV 470 for 4.6 min, UV 450 for 3 min, then UV 325 for rest of run
CHROMATOGRAM

Retention time: 13 Internal standard: retinyl acetate (2.7) Limit of detection: 20 ng/mL
OTHER SUBSTANCES

Extracted: vitamin A (retinol), vitamin E (a-tocopherol), lutein, lycopene, a-carotene, zeaxanthin, trans (3-carotene, 8-tocopherol
KEY WORDS

plasma; serum; protect from sunlight

REFERENCE Bui,M.H. Simple determination of retinol, a-tocopherol and carotenoids (lutein, all-rcms-lycopene, aand p-carotenes) in human plasma by isocratic liquid chromatography, J.Chromatogr.B, 1994, 654, 129-133. SAMPLE

Matrix: blood Sample preparation: 200 |JLL Serum + 200 |xL nonapreno-p-carotene and retinyl butyrate in EtOH, vortex for 10 s, add 1 mL hexane, vortex for 30 s, centrifuge at 1500 g for 5 min. Remove 900 (xL of the hexane layer and evaporate it to a waxy or glassy consistency (not dryness) under vacuum, dissolve in 100 |xL EtOH, add 100 |JLL MeCN, vortex, filter (0.45 jxm), inject a 30 jxL aliquot.
HPLC VARIABLES

Column: 1540 X 4.6 5 jxm Ultramex C18 (Phenomenex) Mobile phase: MeCN:EtOH 50:50 containing 0.1 mIVL diethylamine Column temperature: 29 Flow rate: 0.9 Injection volume: 30 Detector: UV 450
CHROMATOGRAM

Retention time: 8.10 Internal standard: nonapreno-3-carotene (9.5, UV 450), retinyl butyrate (3.5, UV 300) Limit of detection: 13 nM
OTHER SUBSTANCES

Extracted: vitamin A (retinol), vitamin E (a-tocopherol), lutein, zeaxanthin, p-cryptoxanthin, lycopene, a-carotene, retinyl linoleate, retinyl oleate, retinyl palmitate, retinyl stearate
KEYWORDS

serum; use gold fluorescent lamps; hold sample at 4 before injection

REFERENCE Sowell,AL.; Huff,D.L.; Yeager,RR.; Caudill,S.P.; Gunter,E.W. Retinol, a-tocopherol, lutein/zeaxanthin, pcryptoxanthin, lycopene, a-carotene, trans-p-carotene, and four retinyl esters in serum determined simultaneously by reversed-phase HPLC with multiwavelength detection, Clin.Chem., 1994, 40, 411-416. SAMPLE

Matrix: blood Sample preparation: 1 mL Serum + 2.5 mL EtOH, mix for 5 min, add 5 mL n-hexane, mix vigorously, centrifuge at 2000 g for 5 min, repeat extraction with 3 mL n-hexane. Combine the n-hexane layers and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in dichloromethane, inject an aliquot.
HPLC VARIABLES

Guard column: uBondapak C18 Guard-Pak + 50 mm long C18 ODS(4) (Shimadzu) Column: 250 X 4.6 5 |xm Vydac 201 TP 54 C18 Mobile phase: MeOHrMeCN 90:10 (Every 100 injections wash column with MeOHrMeCN: dichloromethane 8:1:1.) Flow rate: 1 Detector: UV 451
CHROMATOGRAM

Retention time: 18.5 (all-trans), 20.7 (9-cis), 21.8 (15-cis), 19.7 (9,15-dicis)

OTHER SUBSTANCES

Extracted: vitamin A (UV 324), vitamin E (UV 291)

KEY WORDS

serum
REFERENCE Ben-Amotz,A. Simultaneous profiling and identification of carotenoids, retinols, and tocopherols by high performance liquid chromatography equipped with three-dimensional photodiode array detection, J.Liq.Chromatogr., 1995, 18, 2813-2825. SAMPLE

Matrix: blood Sample preparation: 200 |xL Serum + 200 |xL 650 ng/mL tocopheryl acetate in MeOH, vortex for 30 s, add 200 |xL n-hexane, shake for 15 min, centrifuge at 3000 rpm for 10 min. Remove 120 |xL of the hexane layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 20 JULL dichloromethane, add 100 jxL MeCN:MeOH 50:50, inject a 50 \xL aliquot.
HPLC VARIABLES

Column: 150 X 4 5 jjim Nucleosil C18 Mobile phase: MeCN:MeOH:dichloromethane 50:45:5 Flow rate: 0.7 Injection volume: 50 Detector: UV 450
CHROMATOGRAM

Retention time: 18.4 (all trans), 19.5 (15,15'-cis) Internal standard: tocopheryl acetate (F ex 295 em 330) (9.3)
OTHER SUBSTANCES

Extracted: vitamin A (F ex 325 em 480), vitamin E (F ex 295 em 330), 7-tocopherol (F ex 295 em 330), retinyl palmitate (F ex 325 em 480), a-carotene
KEYWORDS

serum
REFERENCE Yakushina,L.; Taranova,A. Rapid HPLC simultaneous determination of fat-soluble vitamins, including carotenoids, in human serum, J.Pharm.Biomed.AnaL, 1995, 13, 715-718. SAMPLE

Matrix: blood, tissue Sample preparation: Plasma. 100 u.L Plasma + 100 |xL 0.9% NaCl + 200 uL MeOH, vortex for 30 s, let stand for 10 min, add 400 jxL chloroform, vortex for 4 min. Remove the chloroform layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in MeOH, inject an aliquot. Tissue. Homogenize (liver with Mikro-dismembrator II in liquid nitrogen; other tissue with Ultra-Turrax) tissue with 3 mL 1% acetic acid containing 1 mg/mL ascorbic acid and 10 mM EDTA, add 2 mL MeOH, vortex for 30 s, let stand for 10 min, add 4 mL chloroform, vortex for 4 min. Remove the chloroform layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in MeOH, inject an aliquot.
HPLC VARIABLES

Column: 125 X 4.6 3 jxm Hypersil ODS Mobile phase: MeCN:dichloromethane:MeOH:water 70:10:15:5

Flow rate: 0.5 for 13 min, to 1 over 1 min, maintain at 1 for 10 min, to 1.5 over 1 min, maintain at 1.5 for 21 min, to 2 over 1 min, maintain at 2 for 10 min, return to 0.5 over 1 min, maintain at 0.5 for 2 min. Injection volume: 50 Detector: UV 445
CHROMATOGRAM

Retention time: 39 Limit of detection: 10 ng/mL

OTHER SUBSTANCES

Extracted: vitamin A (UV 350), vitamin E (UV 292)

KEYWORDS

rat; protect from light; liver; plasma; lung

REFERENCE Van Vliet,T.; Van Schaik,R; Van Schoonhoven,J.; Schrijver,J. Determination of several retinoids, carotenoids and E vitamers by high-performance liquid chromatography. Application to plasma and tissues of rats fed a diet rich in either ^-carotene or canthaxanthin, J.Chromatogr., 1991, 553, 179-186. SAMPLE

Matrix: cheese Sample preparation: 500 mg Cheese + 2 mL 60% KOH + 2 mL 95% EtOH + 1 mL 1% NaCl + 5 mL 6% pyrogallol in EtOH, flush tube with nitrogen, seal, heat at 70 for 30 min, cool in ice water, add 15 mL 1% NaCl, extract twice with 15 mL portions of n-hexane: ethyl acetate 90:10. Combine the organic layers and evaporate them to dryness, dissolve the residue in 2 mL mobile phase, inject a 20 yJL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere Si Mobile phase: Gradient. A was n-hexane:isopropanol 99:1. B was n-hexane. A:B 50:50 for 7 min; to 90:10 over 4 min, maintain at 90:10 for 7 min, to 50:50 over 1 min, maintain at 50:50 for 4 min. (About every 100 injections recondition column with 50 mL dichloromethane, 50 mL isopropanol, and 50 mL dichloromethane.) Flow rate: 1.5 Injection volume: 20 Detector: UV 450 ((3-carotene) and F ex 325 em 475 for 3.5 min, ex 280 em 475 for 10.5 min, ex 325 em 475 for 9 min (others)
CHROMATOGRAM

Retention time: 2 Limit of detection: 0.16 ng

OTHER SUBSTANCES

Extracted: vitamin E (a-tocopherol), vitamin A (all-trans-retinol), p-tocopherol, gamma-tocopherol, 8-tocopherol, 13-cis-retinol

KEY WORDS

normal phase; cheese

REFERENCE Panfili,G.; Manzi,R; Pizzoferrato,L. High-performance liquid chromatographic method for the simultaneous determination of tocopherols, carotenes, and retinol and its geometric isomers in Italian cheeses, Analyst, 1994, 119, 1161-1165. SAMPLE Matrix: food

Sample preparation: Dissolve 10 g margarine in 50 mL dichloromethane, add 3 g anhydrous magnesium sulfate, let stand for 2 h with frequent agitation, filter (fritted glass), make up filtrate to 100 mL with dichloromethane, inject a 250 |xL aliquot on to four IxStyragel 100 A GPC columns (Waters) in series, elute with dichloromethane at 1 mL/ min, monitor at 313 nm, collect the fraction corresponding to (3-carotene (at 23.5-26.5 min) and evaporate it to dryness, reconstitute with mobile phase, inject a 100 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jjum Zorbax C18 Mobile phase: MeCN:dichloromethane:MeOH 70:30:0.2 Flow rate: 1 Injection volume: 100 Detector: UV 436 CHROMATOGRAM Retention time: 15 OTHER SUBSTANCES Simultaneous: vitamin A palmitate
KEYWORDS

margarine
REFERENCE Chase,G.W.,Jr.; Akoh,C.C; Eitenmiller,R.R.; Landen,W.O. Liquid chromatographic method for the concurrent analysis of sucrose polyester, vitamin A palmitate, and p-carotene in margarine, J.Liq.Chromatogr., 1995, 18, 3129-3138. SAMPLE

Matrix: fruit, juice, oil, vegetables Sample preparation: Squash, peach. Homogenize (Waring blender) 10-30 g finely chopped squash or peach with 100 mL acetone and 10 g Hyflo supercel for 3 min, filter, repeat extraction of the solids until all the pigment was removed, extract with petroleum ether, wash the organic layer with water, pass it through a short column containing anhydrous sodium sulfate, concentrate under reduced pressure, inject an aliquot. Orange juice, palm tree oil. Homogenize (Waring blender) 10-30 g orange juice or palm tree oil with 100 mL acetone and 10 g Hyflo supercel for 3 min, filter, repeat extraction of the solids until all the pigment was removed, extract with petroleum ether, add 10% KOH in MeOH, let stand overnight, wash the organic layer with water, pass it through a short column containing anhydrous sodium sulfate, concentrate under reduced pressure, inject an aliquot.
HPLC VARIABLES

Column: 250 X 2.2 Ca(OH)2 laboratory packed (Ca(OH)2 from Nakarri Chemicals.) Mobile phase: Isooctane (squash, palm tree oil) or Gradient. Isooctane:acetone from 100:0 to 80:20 over 1 h. (orange juice, peach) Flow rate: 0.5 Injection volume: 10 Detector: UV 450
CHROMATOGRAM

Retention time: 10 (13-cis), 15 (trans), 22 (9-cis) (isocratic mobile phase)

KEYWORDS

squash; peach; orange juice; palm tree oil; normal phase

REFERENCE Carvalho,C.R.L.; Carvalho,P.R.N.; Collins,C.H. High-performance liquid chromatographic determination of the geometrical isomers of p-carotene in several foodstuffs, J.Chromatogr.A, 1995, 697, 289-294.

SAMPLE

Matrix: solutions Sample preparation: Inject a 2-25 |xL aliquot of a solution on mobile phase.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm C30 silica (Anal.Chem. 1994, 66, 1667) Mobile phase: MTBE:MeOH 89:11 Flow rate: 1 Injection volume: 2-25 Detector: UV 453 CHROMATOGRAM Retention time: 50 (all-trans) OTHER SUBSTANCES Simultaneous: isomers
REFERENCE Emenhiser,C; Sander,L.C; Schwartz,S.J. Capability of a polymeric C30 stationary phase to resolve cistrans carotenoid isomers in reversed-phase liquid chromatography, J.Chromatogr.A, 1995, 707, 205216. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 3 |xm Adsorbosphere-HS C18 Mobile phase: MeCN:isopropanol:MeOH 60:30:10 containing 0.1% ammonium acetate Flow rate: 1 Detector: UV 234, UV 295, UV 450 OTHER SUBSTANCES Simultaneous: vitamin E
REFERENCE MaitraJ.; Marcocci,L.; Droy-Lefaix,M.T.; Packer,L. Peroxyl radical scavenging activity ofGingko biloba extract EGb 761, Biochem.PharmacoL, 1995, 49, 1649-1655. SAMPLE

Matrix: vegetables Sample preparation: Macerate vegetables (Waring blender), remove 5 g of this material and homogenize it with 10 mL EtOH (Biohomogenizer) for 3 min, add 2 mL pentane, homogenize for 2 min (purge homogenizer motor with nitrogen), centrifuge at 7000 g for 3 min, remove pentane layer, add 2 g NaCl and 5 mL water to lower layer, shake gently, add 8 mL pentane, shake vigorously for 2 min, centrifuge, remove pentane layer. Combine pentane layers and evaporate (if necessary) under a stream of nitrogen to reduce the volume, make up to 5 or 10 mL with pentane, inject a 20 JJIL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Vydac 201 TP54 C18 Mobile phase: MeOH:MeCN:dichloromethane:hexane 65:27:4:4 Flow rate: 1.5 Injection volume: 20 Detector: UV 450 CHROMATOGRAM Retention time: 14

KEY WORDS

details for SFE also given; squash; broccoli; carrots; collard greens; turnip; kale; mustard greens; zucchini
REFERENCE Marsili,R.; Callahan,D. Comparison of a liquid solvent extraction technique and supercritical fluid extraction for the determination of a- and (3-carotene in vegetables, J.Chromatogr.ScL, 1993, 31, 422428. SAMPLE

Matrix: vegetables Sample preparation: Blanch vegetables in water for 90 s. Stir 20 g homogenized and mashed vegetables with 50 mL ethyl ether for about 4 h, filter, extract the residue again. Combine the filtrates, add 5 mL 20% KOH in MeOH, mix, place in a refrigerator for about 12 h, add 100 mL water, shake vigorously, let stand for about 5 h. Remove the ether layer and concentrate it under vacuum, evaporate traces of ether with a stream of nitrogen, reconstitute with EtOH (?), inject an aliquot.
HPLC VARIABLES Column: 250 X 4.6 5 xm Wakosil-II 5C18 AR polymeric ODS (Wako Chemicals) Mobile phase: THF:MeOH 10:90

Column temperature: 20 Flow rate: 1 Injection volume: 100 Detector: UV 450

CHROMATOGRAM Retention time: 15 OTHER SUBSTANCES

Extracted: a-carotene, p-cryptoxanthin, lutein, lycopene, zeaxanthin

KEYWORDS

carrots; tomatoes; pumpkins

REFERENCE Jinno,K; Lin,Y. Separation of carotenoids by high-performance liquid chromatography with polymeric and monomeric octadecylsilica stationary phases, Chromatographia, 1995, 41, 311-317.

Betahistine
Molecular formula: C8H12N2 Molecular weight: 136.20 CAS Registry No.: 5638-76-6, 5579-84-0 (2HCL) Merck Index: 1224

LednicerNo.: 2 279 SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 3.155 KEYWORDS whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Betaine
Molecular formula: C5H11NO2 Molecular weight: 117.15 CAS Registry No.: 107-43-7, 590-46-5 (HCI), 590-47-6 (monohydrate), 93227-64-6 (sodium aspartate) Merck Index: 1225

SAMPLE

Matrix: solutions Sample preparation: Mix 600 (JLL solution with 60 |xL IS solution, vortex, add a 550 |xL aliquot to a 70 X 5 column of Dowex 1-X8 (OH") form in a Pasteur pipette, elute with 2 mL water, evaporate the eluate to dryness under a stream of air, reconstitute with 100 fiL 2 mM N,N-diisopropylethylamine in MeCN, add 100 |xL 5 mM 4'-bromophenacyl trifluoromethanesulfonate in MecN, vortex for 10 min, add 100 |xL 10 mM hydroxyacetic acid N,N-diisopropylethylammonium salt in MeCN, vortex for 2 min, inject a 15 min aliquot. (Prepare 4'-bromophenacyl trifluoromethanesulfonate as follows. Add 8.8 g p-bromobenzoyl chloride in 40 mL dry ether over 20-30 min to 100 mmoles diazomethane stirred in an ice bath, stir in an ice bath for 8-9 h, let stand at room temperature for 3 h, evaporate the solvent under reduced pressure, recrystallize 4'-bromo-2-diazoacetophenone from ether/hexane (mp 123.5-124 d) (J.Am.Chem.Soc. 1951, 73, 5301). Condense 50 mL anhydrous sulfur dioxide in a flask fitted with a calcium sulfate drying tube, cool in a dry ice/acetone bath, add 2.25 g 4'-bromo-2-diazoacetophenone, stir for 5 min, add 900 IxL anhydrous trifluoromethanesulfonic acid from a freshly opened bottle in one portion, stir for 15 min, remove the cooling bath, after 30 min use an ice/water bath to evaporate the solvent. Dissolve the residue in 100 mL boiling dichloromethane, treat twice with 5 g portions of decolorizing carbon, filter, evaporate the filtrate, recrystallize the residue from pentane:dichloromethane 80:20 to give 4'-bromophenacyl trifluoromethanesulfonate as colorless plates (mp 137-8) (J. Chromatogr. 1984, 299, 365).)
HPLC VARIABLES

Guard column: 50 X 4 Co:Pell ODS Column: 100 X 8 5 |xm Radial-PAK C18 Mobile phase: MeCNrbuffer 70:30 (Prepare by dissolving 70 mg sodium dodecyl sulfate, 140 mg NaH2PO4-H2O, and 300 |xL 3-dimethylamino-l-propanol in 150 mL water, adjusting pH to 6.5 with 85% phosphoric acid, and adding 350 mL MeCN.) Flow rate: 5 Injection volume: 15 Detector: UV 254 CHROMATOGRAM Retention time: 7 Internal standard: (4-bromophenyl)carboxymethyl (6-trimethylammonium)hexanoate (Add an aqueous solution of 6-(trimethylammonium)hexanoic acid (Cl" salt, prepared by methylation of 6-aminohexanoic acid) to a column of Dowex 1-X8 (OH" form), elute with 4 column volumes of water. Evaporate the eluate to dryness, dissolve the residue in DMF, add 1.1 equivalents of triethylamine, add 1.1 equivalents of 2,4'-dibromoacetophenone, stir at 40 for 3 h, add ethyl acetate. Collect the precipitate by filtration and recrystallize it from ethanol/acetone to obtain (4-bromophenyl)carboxymethyl (6-trimethylammonium)hexanoate.) (8) Limit of quantitation: 10 |xM KEYWORDS derivatization

Next Page
REFERENCE Minkler,P.E.; Ingalls,S.T.; Kormos,L.S.; Weir,D.E.; Hoppel,C.L. Determination of carnitine, butyrobetaine, and betaine as 4'-bromophenacyl ester derivatives by high-performance liquid chromatography, J.Chromatogr., 1984, 336, 271-283.

Previous Page

Betamethasone
Molecular formula: C22H29FO5 Molecular weight: 392.47 CAS Registry No.: 378-44-9, 987-24-6 (acetate), 22298-29-9 (benzoate), 5593-20-4 (dipropionate), 151-73-5 (sodium phosphate), 2152-44-5 (17-valerate), 5534-05-4 (acibutate), 360-63-4 (dihydrogen phosphate) Merck Index: 1226 Lednicer No.: 1 198

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) (xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 13.277 KEY WORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: formulations Sample preparation: Condition a 500 mg diol SPE cartridge (Analytichem) with 6 mL dichloromethane. Sonicate a sample of cream containing 366 |xg betamethasone valerate with 5 mL hexane:dichloromethane 70:30 for 5 min, make up to 10 mL with hexane: dichloromethane 70:30, add a 5 mL aliquot to the SPE cartridge, wash with 1 mL hexane: dichloromethane 70:30, elute with two 1 mL portions of MeOH, add 500 /xL 180 jxg/mL hydroxyresorcinol to the eluate, inject a 5 fiL aliquot.

HPLC VARIABLES

Column: 150 X 3.2 5 jxm Hypersil ODS Mobile phase: MeOH:water 75:25 Flow rate: 0.5 Injection volume: 5 Detector: UV 235
CHROMATOGRAM

Retention time: 10 (betamethasone valerate) Internal standard: hydroxyresorcinol OTHER SUBSTANCES Simultaneous: chlorocresol
KEYWORDS

cream; SPE
REFERENCE Di Pietra,A.M.; Andrisano,V.; Gotti,R.; Cavrini,V. On-line post-column photochemical derivatization in liquid chromatographic-diode-array detection analysis of binary drug mixtures, J.Pharm.BiomedAnaL, 1996,14, 1191-1199. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 10 urn Partisil 10 ODS Mobile phase: MeOH:water 75:25 Flow rate: 1.2 Detector: UV 242
CHROMATOGRAM

Retention time: 5 (betamethasone 17-valerate)

REFERENCE Mithani,S.D.; Bakatselou,V.; TenHoor,C.N.; Dressman,J.B. Estimation of the increase in solubility of drugs as a function of bile salt concentration, Pharm.Res., 1996, 13, 163-167. SAMPLE

Matrix: solutions Sample preparation: Pass 20 mL of a solution in water (?) through an Empore C18 SPE disc. Wash with 2.5 mL water, dry, add 1 mL MeOH, let soak for 3 min, elute. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with 1 mL running buffer, inject an aliquot.
HPLC VARIABLES

Column: 150 X 4.6 5 |xm Spherisorb S5 ODS2 Mobile phase: MeOH:water 75:25 Flow rate: 1 Detector: UV 254 OTHER SUBSTANCES Noninterfering: excipients
KEYWORDS

comparison with capillary electrophoresis; SPE

REFERENCE Lucangioli,S.E.; Rodriguez,V.G.; Fernandez Otero,G.C; Vizioli,N.M.; Carducci,C.N. Development and validation of capillary electrophoresis methods for pharmaceutical dissolution assays, J.Capillary Electrophor., 1997, 4, 27-31.

Betaxolol
Molecular formula: C18H29NO3 Molecular weight: 307.43 CAS Registry No.: 63659-18-7, 63659-19-8 (HCI) Merck Index: 1229 LednicerNo.: 4 26 SAMPLE

Matrix: blood Sample preparation: Add 5 mL chloroform:2-propanol:n-heptane 60:14:26, 1.5 mL saturated pH 9.5 ammonium chloride, and 40 jxL 100 |xg/mL prazepam to 2.0 mL postmorten blood. Shake horizontally for 20 min, centrifuge at 2800 g for 15 min. Evaporate organic phase under reduced pressure at 45, dissolve the residue in 100 |xL mobile phase, iriject a 50 jxL aliquot.
HPLCVARIABLES

Column: 300 X 3.9 4 jxm Nova-Pak C18 Mobile phase: MeOH:tetrahydrofuran:100 mM pH 2.6 KH2PO4 65:5:30

Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 220

CHROMATOGRAM

Retention time: 4.32 Internal standard: prazepan (8.81) Limit of quantitation: 100 ng/mL
KEYWORDS

whole blood
REFERENCE BerthaultjR; Kintz,P.; Tracqui,A.; Mangin,P. A fatal case of betaxolol poisoning, J.Anal.ToxicoL, 1997, 21, 228-231. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL 50 mg Bond Elut 40 |xm cyanopropylsilica SPE cartridge with 1 mL MeOH at 6 mL/min and with 1 mL pH 7.4 buffer at 6 mL/min. Centrifuge plasma, add 1 mL plasma at 0.18 mL/min to the SPE cartridge, wash with 1 mL pH 7.4 buffer at 1.5 mL/min, elute with 300 |xL MeOH:2-aminoheptane 99.7:0.3 at 1.5 mL/min, pass 700 |xL pH 3.0 buffer through the cartridge at 1.5 mL/min. Mix both eluates, inject a 250 |xL aliquot. (pH 7.4 Buffer was 250 mL 100 mM KH2PO4 and 195.5 mL 100 mM NaOH, made up to 1 L, if necessary pH adjusted to 7.4. pH 3.0 Buffer was 4 g NaOH in 700 mL water, pH adjusted to 3.0 with 85% phosphoric acid, made up to 1 L with water.)
HPLC VARIABLES

Guard column: 4 X 4 5 jim LiChrospher 100 RP-18 Column: 250 X 4 4 |xm Superspher 100 RP-18 (Merck) Mobile phase: MeCN:buffer 30:70 containing 0.5% 2-aminoheptane (Buffer was 4 g NaOH in 700 mL water, pH adjusted to 3.0 with 85% phosphoric acid, made up to 1 L with water.)

Column temperature: 37

Flow rate: 1.2 Injection volume: 250 Detector: F ex 230 em 300

CHROMATOGRAM Retention time: 15 KEYWORDS

plasma; SPE
REFERENCE Hubert,R; Chiap,R; Moors,M.; Bourguignon,B.; Massart,D.L.; Crommen,J. Knowledge-based system for the automated solid-phase extraction of basic drugs fom plasma coupled with their liquid chromatographic determination. Application to the biodetermination of (3-receptor bocking agents, J.ChromatogrA, 1994, 665, 87-99. SAMPLE

Matrix: blood Sample preparation: 200 (iL Plasma + 50 |xL 50 mM pH 7.4 phosphate buffer + 500 |xL 2% zinc sulfate in MeOH:water 50:50, mix, centrifuge at 13000 rpm for 5 min, inject an aliquot.
HPLC VARIABLES

Guard column: 40 X 4.6 SynChropak bulk support (Knauer) Column: 120 X 4.6 5 fim Spherisorb ODSl C18 Mobile phase: MeCN:MeOH:pH 4.5 acetate buffer (ratio not given) Flow rate: 1 Detector: UV 222
CHROMATOGRAM Retention time: 5.02 OTHER SUBSTANCES

Extracted: cyclopropane carboxylic acid ester prodrug

KEY WORDS

plasma
REFERENCE Hovgaard,L.; Brondsted,H.; Buur,A.; Bundgaard,H. Drug delivery studies in Caco-2 monolayers. Synthesis, hydrolysis, and transport of O-cyclopropane carboxylic acid ester prodrugs of various p-blocking agents, Pharm.Res., 1995, 12, 387-392. SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanol:n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |iL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 |xm NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)

Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 223

CHROMATOGRAM

Retention time: 7.38 Limit of detection: <120 ng/mL

KEY WORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol; aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262. SAMPLE

Matrix: blood, urine Sample preparation: 1 mL Blood or urine + 100 |xL 200 ng/mL (blood) or 90 |xL 1 fxg/mL (urine) cicloprolol hydrochloride in water + 1 mL water + 200 jutL 1 M NaOH, vortex, add 10 mL diethyl ether, shake on a mechanical shaker for 30 min, centrifuge at 1400 g for 15 min. Remove the organic layer and add it to 2 mL 50 mM HCl, mix for 10 min. Remove

the aqueous phase and add it to 200 |L 1 M NaOH, add 10 mL diethyl ether, mix for 10 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40-50, reconstitute the residue in 100 |xL 5 mM N,N-dimethyloctylamine, inject a 90 |JLL (blood) or 20-90 JJLL (urine) aliquot.
HPLC VARIABLES

Column: 75 X 4.5 3 jim Ultrasphere C18 (blood) or 150 X 4.5 5 |xm Ultrasphere C18 (urine) Mobile phase: MeCN:50 mM pH 3.0 N,N-dimethyloctylamine:water 8:10:82 Flow rate: 1 Injection volume: 20-90 Detector: F ex 200 no. 280 emission filter (Schoeffel Model FS 970)
CHROMATOGRAM

Retention time: 9 (blood), 29 (urine) Internal standard: cicloprolol hydrochloride (6 (blood), 20 (urine)) Limit of detection: 10 ng/mL (urine), 1 ng/mL (blood)
OTHER SUBSTANCES

Extracted: metabolites
REFERENCE Wong,Y.W. J.; Ludden,T.M. Determination of pxolol and its metabolites in blood and urine by high-performance liquid chromatography with fluorimetric detection, J.Chromatogr., 1990, 534, 161-172. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) u,L aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 fjim Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 13.405
KEYWORDS

whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 300 X 3.9 5 |xm Nova-Pak C18 Mobile phase: MeOH:buffer 50:50 (Buffer was pH 4.0 phosphate buffer (ionic strength = 0.1) containing 4 mM N,N-dimethyloctylamine, pH readjusted to 4.00 with 85% phosphoric acid.) Column temperature: 30 Flow rate: 1 Injection volume: 100 Detector: UV 220 CHROMATOGRAM Retention time: k' 3.3
OTHERSUBSTANCES

Also analyzed: alprenolol, bopindolol, propranolol, tertatolol

REFERENCE Hamoir,T.; Verlinden,Y.; Massart,D.L. Reversed-phase liquid chromatography of -adrenergic blocking drugs in the presence of a tailing suppressor, J.Chromatogr.ScL, 1994, 32, 14-20. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 |xL aliquot of a 1 mg/mL solution.

HPLC VARIABLES

Column: 250 X 4.6 10 |xm Chiralcel OD Mobile phase: Hexane:isopropanol:diethylamine 80:20:0.1 Flow rate: 0.5 Injection volume: 20 Detector: UV 275
CHROMATOGRAM

Retention time: k' 0.52, 1.57 (enantiomers)

KEYWORDS

chiral
REFERENCE Ekelund,J.; van Arkens,A.; Bronnum-Hansen,K.; Fich,K.; Olsen,L.; Petersen,P.V. Chiral separations of (3-blocking drug substances using chiral stationary phases, J.Chromatogr.A, 1995, 708, 253-261. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 12 jjim l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine chemically bonded to silica (Regis) Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80 Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: k' 9.86

OTHER SUBSTANCES

Also analyzed: acebutolol, alprenolol, antazoline, atenolol, bisoprolol, bopindolol, bupranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cinnarizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, esmolol, famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304
REFERENCE Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on cti-acid glycoprotein as revealed by chemometric analysis of biochromatographic data, Biomed.Chromatogr., 1995, 9, 211-215. SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a solution in 0.5% orthophosphoric acid.
HPLC VARIABLES

Column: 5 mm i.d. 4 |xm Nova-Pak phenyl radial-Pak Mobile phase: MeCN:MeOH:0.05% orthophosphoric acid 24:10:66 Flow rate: 1.6 Detector: F ex 261 em 306
OTHER SUBSTANCES

Simultaneous: dextromethorphan, levorphanol (dextrorphan)

REFERENCE Laslett,T.J.; Alvarez,R; Nation,R.L.; Evans,A.M.; Scott,S.D.; Stupans,! Effect of cyclophosphamide administration on the activity and relative content of hepatic P4502D1 in rat, Xenobiotica, 1995, 25, 1031-1039.

Betotastine
Molecular formula: C21H25CIN2O3 Molecular weight: 388.89 CAS Registry No.: 125602-71 -3

SAMPLE

Matrix: blood, tissue Sample preparation: Blood. Dilute 1 mL plasma with 100 jxL 1 M pH 9.0 phosphate buffer and 100 |xL water, add 8 mL chloroform and extract. (Caution! Chloroform is a carcinogen!) Evaporate the organic layer, dissolve the residue in 400 |xL mobile phase. Inject 200 (JLL aliquot. Tissue. Homogenize the brain with 2-fold the weight of water. Mix 1500 |xL Drain homogenate with 8 mL MeCN, centrifuge, evaporate 8 mL supernatant, dissolve the residue in 1 mL MeCN:water 50:50 and clean it up by a 1 mL 100 Bond Elute C18 SPE cartridge. Evaporate the effluent to dryness, dissolve the residue in 400-500 |xL mobile phase. Inject a 200 |xL aliquot.
HPLC VARIABLES

Column: 250 X 6 Intersil ODS-2 Mobile phase: MeCN:0.018% TFA 17:83 (plasma), MeCN:0.018% TFA 18:82 (tissue) Column temperature: 40 Flow rate: 0.7 Injection volume: 200 Detector: UV 220
CHROMATOGRAM

Limit of quantitation: 10 ng/mL (plasma), 35 ng/mL (brain)

KEYWORDS

brain; cat; mouse; pharmacokinetics; plasma; rat; SPE

REFERENCE Kato,M.; Nishida,A.; Aga,Y.; Kita,J.; Kudo,Y; Narita,H.; Endo,T. Pharmacokinetic and pharmacodynamic evaluation of central effect of the novel antiallergic agent betotastine besilate, Arzneimittelforschung, 1997, 47, 1116-1124.

Bevantolol
Molecular formula: C20H27NO4 Molecular weight: 345.44 CAS Registry No.: 59170-23-9, 42864-78-8 (HCI) Merck Index: 1238 Lednicer No.: 3 28

SAMPLE

Matrix: blood Sample preparation: Condition a 3 mL Bond-Elut C18 SPE cartridge with 1 volume MeCN and 1 volume 25 mM ammonium hydroxide, do not allow to dry. 500 |xL Plasma + 80 fxL water + 1 mL MeCN, vortex for 10 s, centrifuge at 2000 rpm for 10 min, remove the supernatant and add it to 1 mL 25 mM pH 10.5 ammonium hydroxide, vortex, add 100 |xL 250 |xg/mL 2,3,4,5-tetra-O-acetyl-a-D-glucopyranosyl isothiocyanate in MeCN (prepare fresh daily) with vortexing, let stand at room temperature for 10 min, add to SPE cartridge, wash with 1 volume 25 mM ammonium hydroxide, wash with 1 volume water, elute with 1.25 mL MeOH. Evaporate the eluent to dryness under a stream of nitrogen at 45, reconstitute the residue in 500 |xL mobile phase, inject a 75 |xL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 5 jxm Partisil 5 RAC II Mobile phase: MeCN:75 mM (NHJ 2 HPO 4 adjusted to pH 3.5 with phosphoric acid 50:50 Column temperature: 45 Flow rate: 2 Injection volume: 75 Detector: UV 220 CHROMATOGRAM Retention time: 6 (+), 7 (-) Limit of detection: 20 ng/mL Limit of quantitation: 40 ng/mL
KEYWORDS

plasma; chiral; SPE; derivatization

REFERENCE Rose,S.E.; Randinitis,E.J. A high-performance liquid chromatographic assay for the enantiomers of bevantolol in human plasma, Pharm.Res., 1991, 8, 758-762. SAMPLE

Matrix: blood, urine Sample preparation: Plasma. 1 mL Plasma + 1 mL pH 9.5 sodium bicarbonate buffer + 100 ng pronethalol, extract with 5 mL diethyl etherrdichloromethane 60:40. Remove the organic layer and extract it with 200 jxL 100 mM HCl, inject the aqueous phase. Urine. Incubate urine with pH 5.5 acetate buffer and p-glucuronidase at 35, buffer with 1 M pH 9.5 sodium carbonate, extract with diethyl etherrdichloromethane 60:40. Remove the organic layer and extract it with 100 mM HCl, inject the aqueous phase.
HPLC VARIABLES

Column: 5 jxm Spherisorb ODS-2 Mobile phase: MeCN:MeOH:500 mM pH 3.5 phosphate buffer 30:20:50 Injection volume: 200 Detector: F (wavelengths not specified)

CHROMATOGRAM

Retention time: 3.0 Internal standard: pronethalol (plasma), verapamil (urine) Limit of detection: 1000 ng/mL (urine), 50 ng/mL (plasma)
KEYWORDS

plasma; pharmacokinetics
REFERENCE Nattel,S.; Lawand,S.; Matthews,C; McCans,J. Bevantolol disposition in patients with hepatic cirrhosis, J.Clin.PharmacoL, 1987, 27, 962-966.

SAMPLE Matrix: feed Sample preparation: 2 g Feed + 20 mL MeOH, rotate at 20 rpm for 1 h, centrifuge at 1300 rpm for 15 min, inject an aliquot.
HPLC VARIABLES

Guard column: 100 X 6.3 30-38 fjim CorPell ODS (Whatman) Column: 250 X 4.6 10 |xm Lichrosorb RP-18 Mobile phase: MeOH:water:acetic acid 60:40:1 containing 5 mM sodium octanesulfonate (flush column with MeCN:water 50:50 after use) Flow rate: 2 Injection volume: 50 Detector: UV 278
CHROMATOGRAM

Retention time: 10 Limit of detection: 50 |xg/g

KEYWORDS

complexation
REFERENCE Spurlock,C.H.; Schneider,H.G. Liquid chromatographic and ultraviolet spectrophotometric determination of bevantolol and hydrochlorothiazide in feeds, JAssoc.OffAnal.Chem., 1984, 67, 321-324. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 jxL aliquot of a 1 mg/mL solution.

HPLC VARIABLES

Column: 250 X 4.6 10 u-m Chiralcel OD Mobile phase: Hexane:isopropanol:diethylamine 10:90:0.1 Flow rate: 0.5 Injection volume: 20 Detector: UV 275
CHROMATOGRAM

Retention time: k' 0.97, 4.10 (enantiomers)

KEY WORDS

chiral
REFERENCE Ekelund,J.; van Arkens,A.; Bronnum-Hansen,K.; Fich,K.; Olsen,L.; Petersen,P.V. Chiral separations of ^-blocking drug substances using chiral stationary phases, J.ChromatogrA, 1995, 708, 253-261.

Bezafibrate
Molecular formula: C19H20CINO4 Molecular weight: 361.82 CAS Registry No.: 41859-67-0 Merck Index: 1240 Led nicer No.: 3 44

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) \LL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 p,m Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5 CHROMATOGRAM Retention time: 18.268 KEYWORDS whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Bicalutamide
Molecular formula: C18H14F4N2O4S Molecular weight: 430.38 CAS Registry No.: 90357-06-5 Merck Index: 1247

SAMPLE

Matrix: blood Sample preparation: Mix 1 mL plasma, 1 mL 50 mM pH 7.0 phosphate buffer, 6 mL ethyl acetate, and 50 |xL IS in MeOH, centrifuge. Remove a 5 mL portion of the organic layer and evaporate it to dryness. Reconstitute the residue in 400 |xL MeCN:water 30:70, inject a 200 |xL aliquot onto column A, elute with mobile phase A. Collect effluent containing the undifferentiated enantiomers, evaporate to dryness, reconstitute the residue in 400 JiL MeCN:20 mM pH 7.0 phosphate bufferl5:80. Inject a 200 JJLL aliquot onto column B. Elute with mobile phase B.
HPLC VARIABLES

Column: A 100 X 4.6 5 |xm Hypersil ODS; B 5 |xm AGP + 150 X 4.6 Ultron ES-OVM Mobile phase: A MeCN:water 30:70; B MeCN:20 mM pH 7.0 phosphate bufferl5:80 Injection volume: 200 Detector: UV
CHROMATOGRAM

Limit of detection: 5 ng/mL (R), 3.8 ng/mL (S)

KEYWORDS

plasma; pharmacokinetics; chiral

REFERENCE CockshottJ.D.; Oliver,S.D.; Young,J.J.; Cooper,K.J.; Jones,D.C. The effect of food on the pharmacokinetics of the bicalutamide ('casodex') enantiomers, Biopharm.Drug Dispos., 1997, 18, 499-507. SAMPLE

Matrix: tissue Sample preparation: Homogenize tissue in water. Buffer 500 jiL intestinal homogenate with 1.5 mL 50 mM pH 7 phosphate buffer, add 6 mL ethyl acetate, mix, centrifuge. Remove a 5 mL portion of the organic layer and evaporate it to dryness, reconstitute the residue with 500 |xL MeCN:water 50:50. Inject a 50 |xL aliquot.
HPLC VARIABLES

Column: 100 X 4.6 5 fjum Hypersil ODS Mobile phase: MeCN:water 30:70 Injection volume: 50 Detector: UV
KEYWORDS

rat; jejunum; ileum; colon

REFERENCE CockshottJ.D.; Oliver,S.D.; Young,J.J.; Cooper,K.J.; Jones,D.C. The effect of food on the pharmacokinetics of the bicalutamide ('casodex') enantiomers, Biopharm.Drug Dispos., 1997, 18, 499-507.

Bidisomide
Molecular formula: C22H34CIN3O2 Molecular weight: 407.98 CAS Registry No.: 103810-45-3 Merck Index: 1251

SAMPLE

Matrix: blood Sample preparation: Alkalinize plasma with 1 M sodium hydroxide and extract with 1 mL chloroform (Caution! Chloroform is a carcinogen !). Centrifuge, transfer organic layer into tube and evaporate to dryness under a stream of dry nitrogen. Reconstitute samples with 200 jxL mobile phase, inject a 50 fxL aliquot.
HPLC VARIABLES

Column: Brownlee 5 jxm CN Mobile phase: MeCN:20 mM pH 4.0 monosodium phosphate 50:50 Flow rate: 1.0 Injection volume: 50 Detector: UV 207
CHROMATOGRAM

Internal standard: disopyramide Limit of detection: 50 ng/mL

KEYWORDS

plasma; human; dog; pharmacokinetics

REFERENCE Pao,L.-H.; Zhou,S.Y; Cook,C.; Kararli,T.; Kirchhoff,C; Truelove,J.; Karim,A.; Fleisher,D. Reduced systemic availability of an antiarrhythmie drug, bidisomide, with meal co-administration: Relationship with region-dependent intestinal absorption, Pharm.Res., 1998, 15, 221-227.

Bifonazole
Molecular formula: C22H18N2 Molecular weight: 310.40 CAS Registry No.: 60628-96-8 Merck Index: 1260 LednicerNo.: 4 93

SAMPLE

Matrix: formulations Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to about 30 mg, add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of nitrate to 5 mL of 100 [ig/mL ketoconazole in MeOH, make up to 25 mL with MeOH, inject 20 |xL aliquot. Cream. Condition a 500 mg Bond-Elut diol cartridge with 6 mL dichloromethane. Weigh out cream equivalent to about 5 mg of drug, add 30 mL dichloromethane, sonicate for 3 min, make up to 100 mL with dichloromethane, filter. Add a 2 mL aliquot to the cartridge, wash with 2 mL dichloromethanermethanol 4:1, wash with 1 mL MeOH, elute with 3 mL mobile phase. Add eluate to 0.5 mL 100 |xg/mL ketoconazole in MeOH, make up to 5 mL with MeOH, inject 20 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Spherisorb CN Mobile phase: THF:buffer 30:70 (Buffer was 50 mM triethylamine adjusted to pH 3.0 with phosphoric acid.) Flow rate: 1 Injection volume: 20 Detector: UV 230
CHROMATOGRAM

Retention time: 11 Internal standard: ketoconazole (7)

OTHER SUBSTANCES

Simultaneous: clotrimazole, ketoconazole, fenticonazole, tioconazole, isoconazole, econazole, miconazole

KEYWORDS

tablets; creams
REFERENCE Di Pietra,A.M.; Cavrini,V.; Andrisano,V.; Gatti,R. HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations, J.Pharm.Biomed.AnaL, 1992, 10, 873-879. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 62 X 2 packed with chiral packing (Prepare packing by dissolving 4-chloro-3-methylphenylcarbamate cellulose in THF, coat on Nucleosil 1000-7, dry at 60 for 3 h under reduced pressure.) Mobile phase: Hexaneiisopropanol 85:15 Flow rate: 0.1 Injection volume: 20 Detector: UV 254

CHROMATOGRAM Retention time: k' 3.6 KEYWORDS narrow-bore; chiral; a 1.56 REFERENCE Chankvetadze,B-; Chankvetadze,L.; Sidamonidze,S.; Yashima,E.; Okamoto,Y. Enantioseparation of some chiral Pharmaceuticals using narrow-bore liquid chromatography, J.Pharm.Biomed.AnaL, 1995, 13, 695-699.

Bioresmethrin
Molecular formula: C22H26O3 Molecular weight: 338.45 CAS Registry No.: 28434-01-7 Merck Index: 1271

SAMPLE

Matrix: solutions
HPLC VARIABLES

Guard column: 50 X 4 40 |xm pellicular material Column: 250 X 4.6 5 |xm silica (IBM) Mobile phase: Hexane:dichloromethane:isopropanol 99:1:0.07 Flow rate: 1 Injection volume: 10 Detector: UV 254
CHROMATOGRAM

Retention time: k' 6.56 (cis), k' 6.95 (trans)

OTHER SUBSTANCES

Also analyzed: allethrin, chrysanthemol, dimethrin, ethyl chrysanthemate, cyfluthrin (baythroid), permethrin, phenothrin, RU-11679, tetramethrin
KEY WORDS

normal phase
REFERENCE Abidi,S.L. Column selectivity in high-performance liquid chromatography of substituted gem-dimethylcyclopropanes, J.Chromatogr., 1986, 368, 59-76. SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a 0.1-1 mg/mL solution in hexane.
HPLC VARIABLES

Guard column: 5 jim Spherisorb NH2 Column: 250 X 4.6 Pirkle ionic type 1-A column (Technicol) Mobile phase: Hexane:isopropanol 99.95:0.05 Flow rate: 0.8 Detector: UV 230
OTHERSUBSTANCES

Also analyzed: phenothrin, permethrin

KEYWORDS

chiral
REFERENCE Lisseter,S.G.; Hambling,S.G. Chiral high-performance liquid chromatography of synthetic pyrethroid insecticides, J.Chromatogr., 1991, 539, 207-210.

Biotin
Molecular formula: C10H16N2O3S Molecular weight: 244.31 CAS Registry No.: 58-85-5 Merck Index: 1272

SAMPLE

Matrix: blood, formulations Sample preparation: Serum. Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH, 10 mL water, and 5 mL 1% acetic acid in water. 1 mL Serum + 1 mL 10% trichloroacetic acid, mix, centrifuge at 2000 g for 5 min, add a 1.5 mL aliquot of the supernatant to the SPE cartridge, wash with 10 mL 1% acetic acid in water, wash with 1 mL water, elute with 10 mL MeOH. Evaporate the eluate to dryness, reconstitute with 100 |xL MeOH, sonicate, add 100 JULL 1 mg/mL 1-pyrenyldiazomethane in ethyl acetate, heat at 40 for 1 h, cool to room temperature, add 300 |xL MeOH, inject a 10 |xL aliquot. Tablets. Powder tablets, weigh out amount containing 500 |xg biotin, add 30 mL MeOH, sonicate for 10 min, shake for 10 min, centrifuge at 3000 rpm for 5 min, remove the supernatant, repeat the extraction three times. Combine the supernatants and make up to 100 mL with MeOH. Remove a 200 (xL aliquot and add it to 200 uL 5 mg/mL 1-pyrenyldiazomethane (Molecular Probes, Eugene OR) in ethyl acetate, heat at 40 for 1 h, cool to room temperature, add to a Sep-Pak silica SPE cartridge, wash with 5 mL hexane, elute with 10 mL MeOH. Evaporate the eluate to dryness, reconstitute with 1 mL MeOH, inject a 5 jxL aliquot.
HPLC VARIABLES

Column: 150 X 4 5 jjim LiChrosorb Si60 Mobile phase: MeCN:water 43:57 (or 57:43 (?)) Flow rate: 1 Injection volume: 5-10 Detector: F ex 340 em 395, UV 240
CHROMATOGRAM

Retention time: 23 Limit of detection: 100 fmole

KEYWORDS

derivatization; tablets; SPE; serum

REFERENCE Yoshida,T.; Uetake,A.; Nakai,C; Nimura,N.; Kinoshita,T. Liquid chromatographic determination of biotin using 1-pyrenyldiazomethane as a pre-column fluorescent labelling reagent, J.Chromatogr., 1988, 456, 421-426. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 jxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)

HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 jim Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mIVmin) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 8.89 KEYWORDS

whole blood
REFERENCE Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763, 149-163. SAMPLE

Matrix: feed, formulations Sample preparation: Formulations. Dilute 50 JULL liquid vitamin to 2 mL with mobile phase, filter, inject a 20 |xL aliquot. Feed. Extract 100 mg horse feed with 10 mL 1 M NaOH. Remove a 6 mL aliquot and adjust the pH to 6-7 with 1 M HCl, dilute to 100 mL with 100 mM pH 6.0 phosphate buffer, filter, inject a 20 |xL aliquot.
HPLC VARIABLES

Guard column: 15 X 4.6 5 |xm Microsorb C18 Column: 250 X 4.6 5 jjim Microsorb C18 Mobile phase: A was 100 mM pH 6.0 phosphate buffer. B was MeOH:200 mM pH 6.0 phosphate buffer 50:50. A:B was 54:46. Flow rate: 0.4 Injection volume: 20 Detector: F ex 490 em 520 (photon-counting) following post-column reaction. Column effluent mixed with a 2 jxg/mL solution of avidin-FITC (Sigma) in 100 mM pH 7.0 phosphate buffer pumped at 1 mIVmin. The mixture flowed through a 10 m long by 0.5 mm i.d. length of knitted open tubular PTFE tubing to the detector.
CHROMATOGRAM

Retention time: 15 Limit of detection: 4.45 ng/mL

OTHER SUBSTANCES Extracted: biocytin

Noninterfering: DMF, acetone, methyl ethyl ketone, vitamin A, ascorbic acid, vitamin D, vitamin E, pyridoxine, vitamin B12, thiamine, riboflavin, niacin, pantothenic acid
KEYWORDS

liquid vitamin; horse feed; complexation; post-column reaction

REFERENCE Przyjazny,A.; Hentz,N.G.; Bachas,L.G. Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneous fluorophore-linked assay, J.ChromatogrA, 1993, 654, 79-86.

SAMPLE

Matrix: formula Sample preparation: 20 mL Infant formula + 150 jxL concentrated HCl, filter (Whatman No. 1 paper), rinse precipitate with 1 mL water, adjust the pH of the filtrate to 7.0 with 6 M NaOH, extract four times with 8 mL n-hexane, dilute the aqueous layer to 25 mL with water, inject an aliquot.
HPLC VARIABLES

Guard column: 15 X 4.6 5 |mm Microsorb C18 Column: 250 X 4.6 5 jxm Microsorb C18 Mobile phase: MeOH: 100 mM pH 7.0 phosphate buffer 20:80 Flow rate: 0.4 Injection volume: 20 Detector: F ex 495 em 518 following post-column derivatization. The effluent from the column mixed with reagent pumped at 0.1 mL/min and flowed through a 10 m X 0.5 mm i.d. PTFE tube in a knitted open-tubular reactor. The reagent was 2 |xg/mL streptavidinFITC (streptavidin labeled with fluorescein isothiocyanate 1:3.6, Vector Laboratories) in 100 mM pH 9.5 phosphate buffer, prepared fresh daily.) CHROMATOGRAM Retention time: 14 Limit of detection: 97 pg OTHER SUBSTANCES Extracted: biocytin Noninterfering: niacinamide
KEYWORDS

post-column reaction; complexation

REFERENCE Hentz,N.G.; Bachas,L.G. Class-selective detection system for liquid chromatography based on the streptavidin-biotin interaction, Anal.Chem., 1995, 67, 1014-1018. SAMPLE

Matrix: formulations Sample preparation: Powder tablets or capsules, weigh out amount corresponding to 200 |xg biotin, add 30 mL water, sonicate forlO min, make up to 50 mL with water, centrifuge at 2000 g for 10 min, filter (0.45 |xm) the supernatant, inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Tomsorb C18 (Tomsk, Tokyo, Japan) Mobile phase: MeCNrbuffer 5:95 (Buffer was 20 mM sodium dodecyl sulfate adjusted to pH 3.5 with perchloric acid.) Column temperature: 40 Flow rate: 0.5 Injection volume: 20 Detector: F ex 342 em 542 following post-column reaction. The column effluent mixed with 0.03% sodium hypochlorite in 200 mM pH 12.5 borate buffer pumped at 0.3 mL/min and the mixture flowed through a 10 m X 0.5 mm LD. stainless steel tube at 50. The effluent from this tube mixed with the reagent pumped at 0.3 mL/min and the mixture flowed through a 10 m X 0.5 mm LD. stainless steel tube at 50 to the detector. (Prepare the reagent by dissolving 800 mg o-phthalaldehyde in 20 mL EtOH, adding 1.5 mL 3-mercaptopropionic acid and making up to 500 mL with 200 mM pH 10.5 borate buffer.) CHROMATOGRAM Retention time: 27

Limit of detection: 10 ng Limit of quantitation: 20 ng

KEYWORDS

post-column reaction; tablets; capsules; granules

REFERENCE NojirijS.; Kamata,K.; Nishijima,N. Fluorescence detection of biotin using post-column derivatization with OPA in high performance liquid chromatography, J.Pharm.Biomed.Anal., 1998, 16, 1357-1362. SAMPLE

Matrix: formulations Sample preparation: Grind 20 tablets, weight out amount equivalent to 100 jxg biotin, add 60 mL 1.5% phosphoric acid, sonicate for 20 min in a water bath at 50, shake mechanically for 20 min, add 15 mL MeCN, dilute to 100 mL with 1.5% phosphoric afcid, mix. Filter through a 0.45 |xm nylon filter membrane, inject a 150 |xL aliquot.
HPLC VARIABLES

Column: 150 X 4.6 3 jxm YMC Octylsilane C8 (YMC, Wilmington, NC) Mobile phase: MeCNrwater adjusted to pH 2.2 with phosphoric acid 5:95 Column temperature: 50 Flow rate: 2 Injection volume: 150 Detector: UV 200
CHROMATOGRAM

Retention time: 20.5 Limit of quantitation: 500 ng/mL

KEYWORDS

tablets
REFERENCE Ekpe,A.E.; Hazen,C. Liquid chromatographic determination of biotin in multivitamin-multimineral tablets, J.Pharm.Biomed.Anal, 1998, 16, 1311-1315. SAMPLE

Matrix: formulations Sample preparation: Dissolve crushed tablet with sonication in 25 mL pH 3.5 potassium phosphate, centrifuge at 10000 rpm for 5 min, filter (0.45 jim) a 2 mL aliquot. Add filtrate to a C18 Sep-Pak SPE cartridge, wash with 2 mL water, wash with 3 mL phosphate buffer, wash with 2 mL MeCNrphosphate buffer 5:95, elute with MeCNrphosphate buffer 15:85, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.5 5 jim Vydac HS C18 Mobile phase: MeCN:10 mM pH 3.5 KH2PO4 10:90 Detector: UV 230
CHROMATOGRAM

Retention time: 10 Limit of quantitation: 75 |xg/mL

KEYWORDS

tablets; SPE

REFERENCE Hudson,T.S.; Subramanian,S.; Allen,R.J. Determination of pantothenic acid, biotin, and vitamin B12 in nutritional products, J.Assoc.Off.Anal.Chem., 1984, 67, 994-998. SAMPLE

Matrix: formulations
HPLC VARIABLES

Column: 100 X 4 3 jjum Hypersil BDS-C18 Mobile phase: Gradient. MeCNrwater adjusted to pH 2.1 from 0.3:99.7 to 25:75 over 11 min Flow rate: 0.5 Detector: UV 220 CHROMATOGRAM Retention time: 10.2
OTHER SUBSTANCES

Simultaneous: caffeine, citric acid, folic acid, niacin, niacinamide, pantothenic acid, riboflavin, saccharin, thiamine, pyridoxine, vitamin B12, ascorbic acid
KEY WORDS

tablets
REFERENCE Hewlett Packard Leaflet 12-5091-7351 EUS, 1993, 1993, SAMPLE

Matrix: solutions Sample preparation: Evaporate biological samples, add 10 (imoles dibenzo-18-crown-6, add 100 fimoles 2,4'-dibromoacetophenone (p-bromophenacyl bromide), add 25 mg anhydrous potassium carbonate, add 1.6 mL MeCN, reflux for 1 h. For bulk quantities take 55 mg biotin + 5 mL EtOH + 3 mL water, neutralize (phenolphthalein) with 20 mM KOH in MeOH, remove the solvent. Suspend in 5 mL MeCN, add 60 mg potassium carbonate, add 24 mg dibenzo-18-crown-6, add 166 mg 2,4'-dibromoacetophenone, reflux for 1 h.
HPLC VARIABLES

Column: 330 X 4 10 ^m jxBondapak C18 Mobile phase: MeOH:water 60:40 Flow rate: 2 Detector: UV 254
CHROMATOGRAM

Retention time: 17.4 Limit of detection: 10 ng/mL OTHER SUBSTANCES Extracted: dethiobiotin
KEY WORDS

derivatization
REFERENCE Desbene,P.-L.; Coustal,S.; Frappier,F. Separation of biotin and its analogs by high-performance liquid chromatography: convenient labeling for ultraviolet or fluorimetric detection, Anal.Biochem., 1983, 128, 359-362.

SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 150 X 4.6 5 jxm Inertsil ODS-2 Mobile phase: MeCN:50 mM KH2PO4 90:10 Flow rate: 1 Detector: UV 210 CHROMATOGRAM Retention time: 9
OTHER SUBSTANCES

Simultaneous: folic acid, niacin, pantothenic acid, riboflavin, niacinamide

REFERENCE MetaChem Catalog, 1995, p. 21. SAMPLE

Matrix: tissue Sample preparation: Condition two Sep-Pak C18 SPE cartridges with 10 mL MeOH and 10 mL water. 2-3 g Gut tissue or liver + 5 mL 5% trichloroacetic acid + 5 nmole dethiobiotin, homogenize, centrifuge at 10000 g for 15 min, re-extract pellet with 5 mL 5% trichloroacetic acid twice. Combine the supernatants and add them to a SPE cartridge, wash with 10 mL MeCN:water 2:98, elute with 10 mL MeCN:water 15:85, add the eluate to a 70 X 8 column of 200-400 mesh Dowex 1x8 formate, wash with 10 mL water, wash with 10 mL 100 mM potassium formate, elute with 30 mL 100 mM potassium formate. Add the eluate to a SPE cartridge, wash with 10 mL water, elute with 10 mL methyl formate. Evaporate the eluate under a stream of nitrogen, dissolve the residue in 100 (xL 2.5 mM panacyl bromide (p-(9-anthroyl)phenacyl bromide) and 0.5 mM dibenzo-18-crown6 in acetone, add 20-30 mg potassium carbonate, heat at 57 for 3 h, inject an aliquot. (Panacyl bromide is available from Molecular Probes, Eugene OR.)
HPLC VARIABLES

Column: 150 X 4.6 3 |xm Hypersil Mobile phase: Dichloromethane:MeOH 95:5 Flow rate: 1.4 Injection volume: 100 Detector: F ex 380 em 470
CHROMATOGRAM

Retention time: 6.46 Internal standard: dethiobiotin (5.56)

KEYWORDS

SPE; gut; rat; liver; normal phase

REFERENCE Stein,J.; Hahn,A.; Lembcke,B.; Rehner,G. High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide, Anal.Biochem., 1992, 200, 89-94.

Biperiden
Molecular formula: C21H30CINO Molecular weight: 311.47 CAS Registry No.: 514-65-8, 1235-82-1 (HCI), 7085-45-2 (lactate) Merck Index: 1274 Lednicer No.: 1 47

SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 1 mL 1 M pH 10.0 sodium carbonate buffer + 5 mL diethyl ether, shake vigorously for 5 min, centrifuge at 2000 rpm for 5 min. Remove the organic phase and add it to 2 mL 1 M HCl, shake for 2 min, centrifuge at 2000 rpm for 5 min. Remove the aqueous phase and add it to 1 mL 3 M NaOH, add 5 mL diethyl ether, shake for 2 min, centrifuge at 2000 rpm for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in mobile phase, inject an aliquot. (Extraction procedure from Chem. Pharm. Bull. 1985, 33, 4581.)
HPLC VARIABLES

Column: 150 X 3.9 5 |xm Spherisorb C8 Mobile phase: MeCNibuffer 60:40 (Buffer was 1.5 mL triethylamine in 1 L water adjusted to pH 3.0 with 85% phosphoric acid.) Flow rate: 1.5 Detector: UV 199
CHROMATOGRAM Retention time: 5.6 OTHER SUBSTANCES

Simultaneous: hyoscyamine, orphenadrine, benztropine Noninterfering: amantadine, carbidopa, levodopa Interfering: bromocriptine
KEY WORDS

plasma
REFERENCE Selinger,K; Lebel,G.; Hill,H.M.; Discenza,C. High-performance liquid chromatographic method for the analysis of benztropine in human plasma, J.Chromatogr., 1989, 491, 248-252. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 JJIL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 jxm Symmetry C8 (Waters)

Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 14.847 KEYWORDS whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.

Bisacodyl
Molecular formula: C22H19NO4 Molecular weight: 361.40 CAS Registry No.: 603-50-9, 1336-29-4 (complex with tannic acid) Merck Index: 1282

SAMPLE

Matrix: formulations Sample preparation: Tablets. Grind tablets to powder, weigh out an amount equivalent to 5 mg bisacodyl, add 10-15 mL isopropanol, sonicate for 15 min, cool to room temperature, dilute to 25 mL with isopropanol, mix, centrifuge for 5 min. Remove a 5 mL aliquot and evaporate it to dryness under a stream of air, reconstitute the residue in 10 mL mobile phase, inject a 20 ^xL aliquot. Suppositories. Weigh out suppositories equivalent to about 50 mg bisacodyl, dissolve in warm (60) isopropanol, cool to 15, make up to 250 mL with isopropanol, mix, centrifuge an aliquot at 2000 rpm for 5 min. Remove 5 mL supernatant and evaporate it to dryness under a stream of air, dissolve in mobile phase, make up to 10 mL with mobile phase, centrifuge at 2000 rpm for 5 min, inject a 20 fiL aliquot of the lower aqueous phase.
HPLC VARIABLES

Column: 300 X 3.9 10 p,m (xBondapak C18 Mobile phase: MeCN:MeOH:10 mM citric acid 25:25:50 Flow rate: 1.5 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 10 Limit of quantitation: 2500 ng/mL

KEYWORDS

tablets; suppositories
REFERENCE Valenti,L.R; Lau-Cam,C.A. Reverse phase liquid chromatographic determination of bisacodyl in dosage forms, JAssocOffAnaLChem., 1985, 68, 529-532. SAMPLE

Matrix: formulations Sample preparation: Suppositories. Heat a suppository in 150 mL EtOH at 45 until it dissolves, cool to room temperature, make up to 200 mL with EtOH. Remove a 10 mL aliquot and add it to 20 mL mobile phase, filter (GF/C glass microfiber), inject a 20 jxL aliquot. Tablets. Shake 10 tablets in 10 mL buffer until tablets are soft, break up with a glass rod, add 170 mL EtOH, sonicate for 25 min with occasional shaking, cool to room temperature, make up to 200 mL with EtOH. Remove a 10 mL aliquot and add it to 20 mL mobile phase, filter (GF/C glass microfiber), inject a 20 fiL aliquot. Micro-enema. Sonicate micro-enema with 40 mL EtOH for 15 min, cool to room temperature, make up to 50 mL with EtOH. Remove a 10 mL aliquot and add it to 20 mL mobile phase, filter (GF/C glass microfiber), inject a 20 |ULL aliquot. (Buffer was 2.88 g Na2HPO4 and 1.145 g KH2PO4 in 100 mL water.)
HPLC VARIABLES

Column: 250 X 4 5 |xm LiChrospher 60 RP-select B

Mobile phase: MeCN:50 mM KH2PO4 55:45 Flow rate: 1 Injection volume: 20 Detector: UV 214 CHROMATOGRAM Retention time: 7.1 OTHER SUBSTANCES Simultaneous: degradation products KEY WORDS suppositories; tablets; micro-enema REFERENCE Bradshaw,K.M.; Burnett,J.; Sidhu,A.S. High-performance liquid chromatographic determination of bisacodyl in pharmaceutical dosage forms marketed in Australia, J.Pharm.BiomedAnal., 1995, 23, 1355-1362.

Bisantrene
Molecular formula: C22H22N8 Molecular weight: 398.47 CAS Registry No.: 78186-34-2, 71439-68-4 (di HCI) Merck Index: 1284 Led nicer No.: 4 62

SAMPLE

Matrix: bile, blood, urine Sample preparation: Incubate 500 |xL Bile or urine +50 |xL 1 M pH 4.0 sodium acetate + 10 U/mL glucuronidase overnight. 2 mL Plasma or a lesser volume of urine or bile diluted to 1 mL with water + an equal volume of 1 M pH 10 sodium phosphate buffer + 1 |ig IS + 8 mL ethyl acetate, extract. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 300 (xL MeOH with gentle warming, inject a 100 |xL aliquot.
HPLC VARIABLES

Column: 250 mm long 5 |xm LiChrosorb RP-2 C2 Mobile phase: Gradient. MeOH:500 mM pH 5.3 sodium perchlorate from 5:95 to 100:0 over 15 min. (Purify sodium perchlorate solution by passing through a bed of silica gel and activated charcoal before use.) Flow rate: 2 Injection volume: 100 Detector: UV 430 for bisantrene, UV 500 for IS CHROMATOGRAM Retention time: 7 Internal standard: l-[2-(2-hydroxyethyl-l-amino)ethylamino]-4-hydroxy-9,10-anthracenedione Limit of detection: 50 ng/mL (human plasma), 25 ng/mL (rabbit plasma)
KEYWORDS

plasma; rabbit; pharmacokinetics; human

REFERENCE Powis,G. Reversed-phase high-performance liquid chromatographic assay for the antineoplastic agent 9,10-anthracenedicarboxaldehyde bis(4,5-dihydro-lH-imidazol-2-yl hydrazone) dihydrochloride, J.Chromatogr., 1981, 226, 514-520. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 100 |xL 5% perchloric acid, mix, add 100 |xL 25% ammonium hydroxide, add 4 mL ethyl acetate, vortex for 1 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200 JAL 500 mM HCl, inject an aliquot.
HPLC VARIABLES

Column: Reversed-phase C18 (Waters or Bio-Rad) Mobile phase: MeOH:200 mM pH 4.0 ammonium acetate 40:60 Flow rate: 2 Detector: UV 260
CHROMATOGRAM Retention time: 7

Limit of detection: 30 ng/mL

KEYWORDS

plasma
REFERENCE Davis,T.R; Peng,Y.-M.; Goodman,G.E.; Alberts,D.S. HPLC, MS, and pharmacokinetics of melphalan, bisantrene and 13-cis retinoic acid, J.Chromatogr.Sci., 1982, 20, 511-516. SAMPLE

Matrix: blood Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 10 mL MeOH and 5 mL water. Add 1-2 mL plasma to the SPE cartridge, wash with 5 mL water, elute with 300 |xL 500 mM methanolic HCl, inject an aliquot.
HPLC VARIABLES

Guard column: 70 X 2.1 Co:Pell ODS (Whatman) Column: 300 X 3.9 10 |xm jjiBondapak C18 Mobile phase: MeCN:200 mM pH 4.0 ammonium acetate 27:73 Flow rate: 2 Detector: UV 436
CHROMATOGRAM

Retention time: 4 Limit of detection: 10 ng/mL

KEYWORDS

plasma; SPE
REFERENCE Peng,Y.-M.; Ormberg,D.; Alberts,D.S.; Davis,T.P. Improved high-performance liquid chromatography of the new antineoplastic agents bisantrene and mitoxantrone, J.Chromatogr., 1982, 233, 235-247. SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 2 mL chloroform:MeOH:6 M HCl 83:16:1, agitate, centrifuge at 2000 g for 5 min, discard the organic phase, extract the aqueous phase with 3 mL chloroform and 100 JJLL 28% ammonium hydroxide, agitate, centrifuge. Remove 2.5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 |xL mobile phase, agitate, inject a 50-100 |xL aliquot.
HPLC VARIABLES

Column: 300 X 3.9 ixBondapak C18 Mobile phase: MeCN:water:ammonium formate 30:60:5 (v/v/v) Flow rate: 2 Injection volume: 50-100 Detector: UV 260
CHROMATOGRAM

Retention time: 2.5 Internal standard: CL 238,985 (American Cyanamid analog of bisantrene) (4) Limit of detection: 20 ng/mL
KEYWORDS

plasma; pharmacokinetics

REFERENCE Weiss,G.R.; Hersh,M.; Kuhn,J.G.; Ludden,T.M.; von Hoff,D.D.; Kisner,D.L.; Pirtle,T.E.5III A phase I and pharmacokinetic comparison of hepatic arterial and peripheral vein infusions of bisantrene for liver cancer, Cancer Chemother.Pharmacol., 1985, 15, 144-148. SAMPLE

Matrix: blood Sample preparation: Adjust pH of 1 mL plasma to 11 with 50 |xL 1 M NaOH, add 5 mL dichloromethane, extract. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 250 |xL mobile phase, centrifuge at 15600 g for 1 min, inject a 150 jxL aliquot of the supernatant.
HPLC VARIABLES

Guard column: 37-50 |xm C18/Corasil Column: 300 X 3.9 10 jxm ixBondapak C18 Mobile phase: MeCN:80 mM pH 3.0 sodium formate 28:72 Flow rate: 1 Injection volume: 150 Detector: E, BAS LC-4B detector, TL-5 glassy carbon electrode at +0.75 V, Ag/AgCl reference electrode
CHROMATOGRAM

Retention time: 8 Internal standard: bisantrene OTHER SUBSTANCES Extracted: mitoxantrone

KEY WORDS

plasma; pharmacokinetics; bisantrene is IS

REFERENCE Choi,K.E.; Sinkule,J.A.; Han,D.S.; McGrath,S.C.; Daly,K.M.; Larson,RA. High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection, J.Chromatogr., 1987, 420, 81-88. SAMPLE

Matrix: blood, urine Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 4 mL MeOH, 4 mL MeOH:water 50:50, and 10 mL 50 mM 50 mM sodium phosphate. Add 3 mL plasma or urine to the SPE cartridge, wash with 4 mL 50 mM sodium phosphate, elute with 6 mL chlorofornr.MeOH 2:1. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 50 |xL N,N-dimethylacetamide and 250 u.L saline, centrifuge at 12000 g for 15 min, inject an aliquot of the supernatant.
HPLC VARIABLES

Column: 300 X 4 10 jim ixBondapak C18 Mobile phase: MeOH:water 40:60 containing 20 mM ammonium acetate, pH 4.0 Flow rate: 1.5 Detector: F ex 260 em 550
CHROMATOGRAM

Retention time: 8.5 Limit of detection: 2 ng/mL

KEY WORDS

plasma; SPE

REFERENCE Lu,K; Savaraj,N.; Huang,M.T.; Moore,D.; Loo,T.L. High performance liquid chromatography (HPLC) of the new antineoplastic 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-lH-imidazole-2yDhydrazone] dihydrochloride (CL 216,942; bisantrene), J.Liq.Chromatogr., 1982, 5, 1323-1328.

Bisoprolol
Molecular formula: C18H31NO4 Molecular weight: 325.45 CAS Registry No.: 66722-44-9,104344-23-2 (fumarate) Merck Index: 1336 Lednicer No.: 4 28 SAMPLE

Matrix: blood Sample preparation: 1 mL Plasma + 200 |xL pH 9.2 bicine (N,N-bis(2-hydroxyethyl)glycine) + 4 mL ethyl acetate, shake vigorously for 5 min, centrifuge at 2000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 60, reconstitute the residue in 200 |xL MeOH, inject an aliquot of 100 |JLL or less.
HPLCVARIABLES

Guard column: 10 mm long LiChrosorb CN Column: 250 X 4 10 jxm LiChrosorb CN Mobile phase: MeOH:isopropanol:1.16 M perchloric acid 75:25:0.5 Flow rate: 2.5 Injection volume: =^100 Detector: F ex 235 em 310 CHROMATOGRAM Retention time: 3.6 Internal standard: bisoprolol OTHER SUBSTANCES Extracted: sotalol Simultaneous: verapamil Noninterfering: acebutolol, amiodarone, disopyramide, propafenone, quinidine
KEY WORDS

hydroquinidine,

plasma; bisoprolol is IS; pharmacokinetics

REFERENCE Poirier,J.-M.; Lebot,M.; Cheymol,G. Rapid and sensitive column liquid chromatographic determination of sotalol in plasma, J.Chromatogr., 1989, 493, 409-413. SAMPLE

Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform: isopropanolin-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES

Column: 300 X 3.9 4 jjum NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)

Column temperature: 30

Flow rate: 0.8 Injection volume: 50 Detector: UV 225

CHROMATOGRAM

Retention time: 5.84 Limit of detection: <120 ng/mL

KEY WORDS

whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; najorphine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; alminoprofen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol; aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen; dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40, 254-262. SAMPLE

Matrix: blood, urine Sample preparation: Condition a Bond Elut SI SPE cartridge with 3 mL chloroform. 1 m l Plasma or 500 |xL urine + 200 (plasma) or 500 (urine) jxL water + 200 (plasma) or 300 (urine) JULL 100 mM NaOH + 7 mL chloroform, shake for 10 min, centrifuge at 1800 g for 5 min. Remove the organic layer and add it to the SPE cartridge, wash with 1 mL MeOH, elute with 1 mL MeOHrtriethylamine 95:5. Evaporate the eluate to dryness under

a stream of nitrogen at 60, reconstitute the residue in 200 jxL IS solution, inject a 100 IxL aliquot. (Prepare IS solution by repeatedly injecting high concentrations of IS solution onto this HPLC system, collect the third peak, evaporate the collected eluates to dryness under a stream of nitrogen, reconstitute with mobile phase to an appropriate concentration.)
HPLC VARIABLES

Guard column: 50 X 4.6 10 jim Chiralcel OD (Daicel) Column: 250 X 4.6 10 jxm Chiralcel OD (Daicel) Mobile phase: Hexane:isopropanol 100:9 containing 0.01% diethylamine Column temperature: 25 Flow rate: 0.5 Injection volume: 100 Detector: F ex 228 em 298
CHROMATOGRAM

Retention time: 20 (R(+)), 30 (S(-)) Internal standard: l-[p-(tetrahydro-3-furanyl)phenoxy]-3-isopropylamino-2-propanol (37) Limit of detection: 2 ng/mL
KEYWORDS

plasma; chiral; SPE

REFERENCE Suzuki,T.; Horikiri,Y.; Mizobe,M.; Noda,K. Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans, J.Chromatogr., 1993, 619, 267-273. SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 (xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 200.5
CHROMATOGRAM Retention time: 12.283 KEY WORDS

whole blood

REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163. SAMPLE

Matrix: bulk Sample preparation: Dissolve in mobile phase, inject a 20 jxL aliquot.
HPLC VARIABLES

Column: 250 X 4 10 |xm LiChrosorb RP-18 Mobile phase: MeCNrbuffer 50:50 (Buffer was 50 mM (NHJ 2 HPO 4 adjusted to pH 7.0 with 5 M orthophosphoric acid.) Flow rate: 1.5 Injection volume: 20 Detector: UV 226
CHROMATOGRAM

Retention time: 7 Limit of detection: 5-20 ng

OTHER SUBSTANCES

Simultaneous: fumaric acid, impurities

REFERENCE Agapova,N.N.; Vasileva,E. High-performance liquid chromatographic method for the determination of bisoprolol and potential impurities, J.ChromatogrA, 1993, 654, 299-302. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 300 X 3.9 5 jim Nova-Pak C18 Mobile phase: MeOH:buffer 40:60 (Buffer was pH 4.0 phosphate buffer (ionic strength = 0.1) containing 3.33 mM N,N-dimethyloctylamine, pH readjusted to 4.00 with 85% phosphoric acid.) Column temperature: 30 Flow rate: 1 Injection volume: 100 Detector: UV 220 CHROMATOGRAM Retention time: k' 3.85
OTHER SUBSTANCES

Also analyzed: carvedilol, labetalol, metipranolol, oxprenolol, talinolol, toliprolol

REFERENCE Hamoir,T.; Verlinden,Y.; Massart,D.L. Reversed-phase liquid chromatography of p-adrenergic blocking drugs in the presence of a tailing suppressor, J.Chromatogr.ScL, 1994, 32, 14-20. SAMPLE

Matrix: solutions Sample preparation: Inject a 20 |xL aliquot of a 1 mg/mL solution.

HPLC VARIABLES

Column: 250 X 4.6 10 u.m Chiralcel OD

Mobile phase: Hexane:isopropanol:diethylamine 80:20:0.1 Flow rate: 0.5 Injection volume: 20 Detector: UV 275
CHROMATOGRAM

Retention time: k' 0.69, 1.38 (enantiomers)

KEYWORDS

chiral
REFERENCE Ekelund,J.; van Arkens,A.; Bronnum-Hansen,K.; Fich,K; Olsen,L.; Petersen,RV. Chiral separations of (3-blocking drug substances using chiral stationary phases, J.ChromatognA, 1995, 708, 253-261. SAMPLE Matrix: solutions HPLC VARIABLES

Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine chemically bonded to silica (Regis) Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80 Flow rate: 1 Detector: UV 254
CHROMATOGRAM Retention time: k' 4.43 OTHER SUBSTANCES

Also analyzed: acebutolol, alprenolol, antazoline, atenolol, betaxolol, bopindolol, bupranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cinnarizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, esmolol, famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304
REFERENCE Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on cti-acid glycoprotein as revealed by chemometric analysis of biochromatographic data, Biomed.Chromatogr., 1995, 9, 211-215.

Bitolterol
Molecular formula: C28H31NO5 Molecular weight: 461.56 CAS Registry No.: 30392-40-6, 30392-41-7 (mesylate) Merck Index: 1344 LednicerNo.: 3 22

SAMPLE

Matrix: formulations Sample preparation: Discharge aerosol into 10 mL MeOH in a 50 mL beaker until about 1.2 g sample is collected, rinse aerosol outlet with MeOH, determine weight of sample by difference in weight of can, dilute with MeCN:water:acetic acid 60:38:2 to about 20 jig/ mL bitolterol, inject an aliquot.
HPLC VARIABLES

Column: 250 X 4.6 10 |xm Partisil ODS-3 Mobile phase: MeCN:water:acetic acid:sodium octanesulfonate 600:380:20:0.65 (v/v/v/w) Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 8
OTHER SUBSTANCES

Simultaneous: colterol, degradation products

KEY WORDS

aerosols
REFERENCE Wilson,T.D.; Fogarty,D.F. The effect of column age on system suitability parameters for an HPLC assay of bitolterol mesylate aerosols, J.Chromatogr.ScL, 1988, 26, 60-66. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 250 X 4.6 10 |xm Partisil ODS-3 Mobile phase: MeCN:water:glacial acetic acid:sodium octanesulfonate 600:380:20:0.65 Flow rate: 1 Injection volume: 20 Detector: UV 254 CHROMATOGRAM Retention time: 7 (mesylate) OTHER SUBSTANCES Simultaneous: impurities, colterol
KEYWORDS

stability-indicating

REFERENCE Wilson,T.D. High-performance liquid chromatographic determination of Tornalate in solution dosage forms; a specificity study, J.Chromatogr., 1987, 391, 409-418.

Bleomycin sulfate
Molecular formula: C 55 H 84 N 17 O 21 S 3 (bleomycin A 2 )

Molecular weight: 1415.59 CAS Registry No.: 9041-93-4, 11056-06-7 (free base), 58995-26-9 (bleomycin A1), 11116-31-7 (bleomycin A2), 11116-32-8 (bleomycin A5), 37293-17-7 (bleomycin A6), 41138-54-9 (bleomycin B1), 9060-10-0 (bleomycin B2), 9060-11-1 (bleomycin B4), 73666-80-5 (bleomycin B6) Merck Index: 1351

SAMPLE

Matrix: blood, hepatocytes Sample preparation: 300 |xL Plasma or hepatocyte suspension + 0.9 jig peplomycin + 75 IxL trichloroacetic acid:water 20:80 containing 1 mM copper sulfate, vortex for 1 min, centrifuge at 850 g for 10 min, rewash the pellet with 75 |xL trichloroacetic acid:water 20:80 containing 1 mM copper sulfate, vortex for 1 min, centrifuge at 850 g for 10 min. Combine the supernatants, make up to 300 jxL with water, inject a 100 |xL aliquot.
HPLC VARIABLES

Guard column: 37 X 3.9 Corasil C18 Column: 250 X 10 7 |xm Lichrosorb RP-18 Mobile phase: Gradient. A was 5 mM sodium pentanesulfonate in 0.5% acetic acid, adjusted to pH 4.3 with 28% ammonia. B was MeOH:MeCN 25:75 containing 5 mM sodium pentanesulfonate and 0.5% acetic acid. A:B from 87:13 to 67:33 over 20 min Column temperature: 35 Flow rate: 1.8 Injection volume: 100 Detector: F ex 297 em 355
CHROMATOGRAM

Retention time: 7 (deamidobleomycin A2), 8 (bleomycin A2), 9 (deamidobleomycin B2), 10 (bleomycin B2) Internal standard: peplomycin (14.5) Limit of detection: 70 ng/mL

KEY WORDS

plasma; pharmacokinetics
REFERENCE Mahdadi,R.; Kenani,A.; Pommery,N.; Pommery,J.; Henichart,J.R; Lhermitte,M. High-performance liquid chromatography assay of bleomycin in human plasma and rat hepatocytes in culture, Cancer Chemother.PharmacoL, 1991, 28, 22-26. SAMPLE

Matrix: reaction mixtures Sample preparation: If necessary, remove oxidizing power of solution by adding sodium metabisulfite, inject a 20 jxL aliquot.
HPLC VARIABLES

Guard column: 15 X 4.6 5 |xm Microsorb C8 Column: 250 x 4.6 5 |xm Microsorb C8 Mobile phase: MeCN:5.5 mM sodium octanesulfonate + 20 mM trisodium citrate dihydrate adjusted to pH 3 with concentrated HCl 25:75 Flow rate: 1 Injection volume: 20 Detector: UV 230
CHROMATOGRAM

Retention time: 6.2 Limit of detection: 10000 ng/mL

REFERENCE Lunn,G.; Rhodes,S.W.; Sansone,E.B.; Schmuff,N.R. Photolytic destruction and polymeric resin decontamination of aqueous solutions of Pharmaceuticals, J.Pharm.Sci., 1994, 83, 1289-1293. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 300 X 4.6 jxBondapak C18 Mobile phase: Gradient. All solvents contained 5 mM ammonium formate. MeOH:water 15:85 for 2 h, to 30:70 over 1 h, to 95:5 over 1.5 h. Flow rate: 1.5 Detector: UV 254
CHROMATOGRAM

Retention time: 80 (bleomycin A2), 150 (bleomycin B2), 200 (bleomycin Al), 240 demethylbleomycin A2)
REFERENCE Rzeszotarski,W.J.; Eckelman,W.C; Reba,R.C. Reversed-phase high-performance liquid chromatography of bleomycin, J.Chromatogr., 1976, 124, 88-91. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 300 X 4.6 fjiBondapak C18 Mobile phase: MeOH:water 50:50 containing 5 mM heptanesulfonic acid, adjust pH to 8.3 with concentrated ammonium hydroxide Flow rate: 1.5 Detector: UV 290

CHROMATOGRAM

Retention time: 6 (bleomycin B2), 12 (bleomycin A2) Limit of detection: 10 pmole

REFERENCE Sakai,T.T. Paired-ion high-performance liquid chromatography of bleomycins, J.Chromatogr., 1978, 161, 389-392. SAMPLE

Matrix: solutions Sample preparation: Dissolve in water, add a slight molar excess of copper sulfate, inject a 10-20 |xL aliquot.
HPLC VARIABLES

Guard column: 23 X 3.9 30-38 |xm Corasil C18 Column: 300 X 3.9 10 jxm jxBondapak C18 Mobile phase: Gradient. A was 5 mM sodium pentanesulfonate in 0.5% glacial acetic acid in water, adjusted to pH 4.3 with concentrated ammonium hydroxide. B was MeOH containing 5 mM sodium pentanesulfonate and 0.5% glacial acetic acid. A:B from 72:28 to 52:48 over 45 min. Flow rate: 1.5 Injection volume: 10-20 Detector: UV 280
CHROMATOGRAM

Retention time: 5 (bleomycinic acid), 10 (bleomycin B'l), 11.5 (bleomycin A2), 12.5 (bleomycin A5), 17.5 (bleomycin B2), 22.5 (bleomycin B4), 25.5 (bleomycin B6), 29 (bleomycin CHP), 32 (bleomycin PEPP), 35 (demethylbleomycin A2) Limit of detection: 50 pmole
KEY WORDS

chelates
REFERENCE Klett,R.P.; Chovan,J.P.; Danse,I.H.R. Reversed-phase paired-ion high-performance liquid chromatographic method for the separation and quantification of multiple bleomycin congeners, J.Chromatogr., 1984, 310, 361-371. SAMPLE

Matrix: solutions Sample preparation: Dissolve in water, add a slight molar excess of copper sulfate, inject a 10-20 |xL aliquot.
HPLC VARIABLES

Guard column: 23 X 3.9 30-38 [Lm Corasil C18 Column: 150 X 3.9 4 |xm Novapak C18 Mobile phase: Gradient. A was 5 mM sodium heptanesulfonate in 0.5% glacial acetic acid in water, adjusted to pH 4.3 with concentrated ammonium hydroxide. B was MeOH containing 5 mM sodium heptanesulfonate and 0.5% glacial acetic acid. A:B from 40:60 to 50:50 over 30 min. Flow rate: 0.7 Injection volume: 10-20 Detector: UV 280

CHROMATOGRAM

Retention time: 5 (epibleomycin A2), 8 (epibleomycin B2), 11 (desamidobleomycin A2), 12 (bleomycin A2), 14 (isobleomycin A2), 18 (desamidobleomycin B2), 20 (bleomycin B2), 21 (isobleomycin B2), 22 (epidemethylbleomycin A2), 24 (desamidodemethylbleomycin A2), 25.5 (demethylbleomycin A2), 28 (isodemethylbleomycin A2)
KEY WORDS

chelates
REFERENCE Klett,R.R; Chovan,J.P. Modification of a new high-performance liquid chromatographic method for bleomycin to separate epi-, iso-, desamido-, and unmodified analogs, J.Chromatogr., 1985, 337, 182-186. SAMPLE

Matrix: solutions
HPLC VARIABLES

Column: 50 X 5 10 |xm Mono S HR 5/5 cation-exchange (Pharmacia LKB) Mobile phase: Gradient. A was 50 mM pH 6.5 ammonium formate. B was 1 M pH 6.5 ammonium formate. A:B from 98:2 to 95:5 over 30 min, to 75:25 over 20 min, to 0:100 over 10 min. Column temperature: 4 Flow rate: 1 Detector: UV 280
CHROMATOGRAM

Retention time: 12 (bleomycin A2), 41 (bleomycin B2)

REFERENCE Mistry,J.S.; Sebti,S.M.; Lazo,J.S. Separation of bleomycins and their deamido metabolites by high-performance cation-exchange chromatography, J.Chromatogr., 1990, 514, 86-90. SAMPLE

Matrix: solutions Sample preparation: Make up a 1 mg/mL (1 U/mL) aqueous solution, inject a 20 JJLL aliquot.
HPLC VARIABLES

Guard column: 4 x 4 5 (xm LiChrospher RP-Select B Column: 125 X 4 5 fxm LiChrospher RP-Select B Mobile phase: Gradient. A was 10 mM sodium perchlorate in 0.1% aqueous phosphoric acid. B was MeCN. A:B from 95:5 to 75:25 over 13 min, to 0:100 over 1 min, maintain at 0:100 for 1 min, re-equilibrate at initial conditions for 5 min. Flow rate: 1.5 Injection volume: 20 Detector: UV 240
CHROMATOGRAM

Retention time: 6.5 (bleomycin A2), 7.8 (bleomycin B2), 12.3 (demethylbleomycin A2) Limit of detection: 10 fxg/mL
REFERENCE Fiedler,H.-R; Wachter,J. High-performance liquid chromatographic determination of bleomycins, J.Chromatogr., 1991, 536, 343-347.

Boldenone
Molecular formula: C19H26O2 Molecular weight: 286.41 CAS Registry No.: 846-48-0, 13103-34-9 (undecylenate) Merck Index: 1354 Led nicer No.: 2 153

SAMPLE

Matrix: formulations Sample preparation: Oils. 1 mL Sample + 25 mL MeOH:water 90:10, shake vigorously for 5 min, centrifuge, inject a 10 jxL aliquot of the supernatant. Tablets. Grind a tablet to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aquedus). Make up 5 mL to 50 mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Zorbax ODS Mobile phase: MeOH:water 75:25 Flow rate: 1.5 Injection volume: 10 Detector: UV 240
CHROMATOGRAM

Retention time: 4.8 Limit of detection: 5 |xg/mL

OTHER SUBSTANCES

Simultaneous: methandrostenolone, nandrolone, norgestrel, testosterone, dehydroepiandrosterone (UV 210), mibolerone, methyltestosterone, methandriol (UV 210), norethindrone acetate, calusterone, mesterolone (UV 210), norethandrolone, trenbolone acetate, benzyl benzoate, nandrolone acetate, testosterone acetate, stanozolol, oxymetholone, nandrolone propionate, methenolone acetate, testosterone propionate, aspirin, caffeine, formebolone, benzyl alcohol, testolactone, cortisone Interfering: fluoxymesterone, norethindrone, oxandrolone (UV 210), ethisterone
KEYWORDS

oils; tablets; suspensions

REFERENCE Walters,M.J.; Ayers,R.J.; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry, JAssoc.OffAnalChem., 1990, 73, 904-926. SAMPLE

Matrix: formulations Sample preparation: Oils. 1 mL Sample + 25 mL MeOH:water 90:10, shake vigorously for 5 min, centrifuge, inject a 10 jxL aliquot of the supernatant. Tablets. Grind a tablet to a fine powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 fim), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous). Make up 5 mL to 50 mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 (JLL aliquot of the remaining filtrate.
HPLC VARIABLES

Column: 250 X 4.6 5 |xm Zorbax ODS

Mobile phase: MeOH Flow rate: 1.5 Injection volume: 10 Detector: UV 240
CHROMATOGRAM

Retention time: 4.5 (boldenone undecylenate) Limit of detection: 5 |xg/mL

OTHER SUBSTANCES

Simultaneous: testosterone undecenoate, nandrolone decanoate, methandriol dipropionate, testosterone decanoate, nandrolone laurate, testosterone undecanoate, testosterone, methenolone acetate, testosterone propionate, nandrolone phenylpropionate, testosterone phenylpropionate, testosterone isocaproate, trenbolone hexahydrobenzylcarbonate Interfering: testosterone enanthate, methenolone enanthenate, testosterone cypionate
KEY WORDS

oils; tablets; suspensions

REFERENCE Walters,M.J.; Ayers,R.J.; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry, JAssoc.OffAnal.Chem., 1990, 73, 904-926. SAMPLE

Matrix: formulations Sample preparation: Crush tablets, weigh out amount equivalent to 10 mg steroid, dissolve in 10 mL MeOH, sonicate for 15 min, filter. 1 mL Filtrate + 5 mL MeOH + 4 mL water, inject a 25 |xL aliquot.
HPLC VARIABLES

Column: 250 X 4.6 5 jjum Zorbax ODS Mobile phase: Gradient. MeOH:water from 70:30 to 100:0 over 15 min, maintain at 100:0 for 15 min. Flow rate: 1 Injection volume: 25 Detector: UV 240
CHROMATOGRAM

Retention time: 6.8 (boldenone), 13.4 (boldenone acetate), 25.1 (boldenone undecylenate)
OTHER SUBSTANCES

Simultaneous: clostebol acetate, danazol (UV 280), fluoxymesterone, methandriol, methandriol-3-acetate, methandriol dipropionate, methandrostenolone, methyltestosterone, nandrolone, nandrolone decanoate, nandrolone phenylpropionate, nandrolone propionate, stanolone, stanozolol, testosterone, testosterone acetate, testosterone cypionate, testosterone enanthate, testosterone isobutyrate, testosterone propionate, testosterone undecanoate Noninterfering: oxandrolone, oxymetholone, testosterone decanoate, testosterone isocaproate
KEY WORDS

tablets
REFERENCE Lurie,I.S.; Sperling,A.R.; Meyers,R.P. The determination of anabolic steroids by MECC, gradient HPLC, and capillary GC, J.Forensic ScL, 1994, 39, 74-85.

SAMPLE

Matrix: solutions Sample preparation: Dissolve in MeOH at a concentration of 100 |xg/mL, inject a 5 fxL aliquot.
HPLC VARIABLES

Guard column: 70 X 2.1 COrPeIl ODS Column: 300 X 3.9 Bondex C18 (Phenomenex) Mobile phase: MeOHrwater 75:25 Flow rate: 1 Injection volume: 5 Detector: UV 254
CHROMATOGRAM

Retention time: 3.5 (boldenone), 5.5 (boldenone acetate), 16 (boldenone benzoate)

OTHER SUBSTANCES

Also analyzed: nandrolone, nandrolone propionate, nandrolone phenylpropionate

REFERENCE Noggle,ET.,Jr.; Clark,C.R.; DeRuiter,J. Liquid chromatographic and mass spectral analysis of the anabolic 17-hydroxy steroid esters, J.Chromatogr.ScL, 1990, 28, 263-268. SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a 100 jjig/mL solution in MeOH.
HPLC VARIABLES

Guard column: 70 X 2.1 Whatman CO:Pell ODS Column: 300 X 3.9 Bondex C18 Mobile phase: MeOH:water 70:30 Flow rate: 1 Injection volume: 5 Detector: UV 254
CHROMATOGRAM Retention time: 6 OTHER SUBSTANCES

Simultaneous: methyltestosterone, nandrolone, methandrostenolone, testosterone, danazol, fluoxymesterone

REFERENCE Noggle,F.T.,Jr.; Clark,C.R.; DeRuiter,J. Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids, J.Chromatogr.ScL, 1990, 28, 162-166. SAMPLE

Matrix: solutions Sample preparation: Inject a 5 ^xL aliquot of a 10 jxg/mL solution in MeOH.
HPLCVARIABLES

Column: 75 X 4.6 3 jim Ultrasphere ODS Mobile phase: MeCNrIO mM ammonium acetate buffer 45:55 Flow rate: 0.5 Injection volume: 5 Detector: UV 254

CHROMATOGRAM Retention time: 4.091 OTHER SUBSTANCES

Simultaneous: epimethandienone, epitestosterone, fluoxymesterone, 6p-hydroxymethandienone, methandienone, norethindrone, oxymetholone (UV 280), trenbolone
REFERENCE Barr6n,D.; Pascual,J.A.; Segura,J.; Barbosa,J. Prediction of LC retention of steroids using solvatochromic parameters, Chromatographia, 1995, 41, 573-580. SAMPLE

Matrix: urine Sample preparation: Condition a 200 mg 40 |jim C18 SPE cartridge (J.T.Baker model 7020-2) with 2 mL MeOH and 1 mL 25 mM pH 5.5 ammonium acetate (A). Condition a 200 mg 40 jjim C18 SPE cartridge (J.T.Baker model 7020-2) with 2 mL MeOH and 1 mL water (B). 2 mL Urine + 2 mL MeOH:650 mM pH 5.4 ammonium acetate 20:80 + 75 |JLL 25.3 |xM 19-nortestosterone sodium sulfate in water, sonicate for 15 min, centrifuge at 1000 g for 5 min, add to SPE cartridge (A), wash with 1 mL 25 mM pH 5.5 ammonium acetate, wash with 2 mL MeOH:25 mM pH 5.5 ammonium acetate 40:60, wash with 3 mL water, elute with 2 mL MeOH:water 35:65. Add 2 mL MeOH to the eluate and evaporate it under a stream of nitrogen at 60, dissolve the residue in 100 |xL MeOH, add 5 mL ethyl acetate saturated with 2.2 M sulfuric acid (one tenth volume), heat at 50 for 50 min, cool, wash twice with 2 mL 940 mM pH 10.3 sodium carbonate (pH adjusted with sodium bicarbonate), wash with 3 mL water. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 60, transfer the residue with three 200 fxL portions of MeOH:water 50:50 to SPE cartridge (B), wash with 1 mL water, wash with 2 mL MeOH:water 55:45, elute with 2 mL MeOH:water 80:20. Evaporate the eluate to dryness under a stream of nitrogen at 60, reconstitute the residue in 100 jxL mobile phase, inject a 20 |xL aliquot. (Boldenone sulfate, isolated using SPE cartridge (A), is solvolysed to boldenone which is purified using SPE cartridge (B).)
HPLC VARIABLES

Column: 83 X 4.6 3 |xm Pecosphere-3C C18 Mobile phase: MeCN:MeOH:25 mM pH 5.5 ammonium acetate 7:50:43 Flow rate: 1.1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM Retention time: 6.2

Internal standard: 19-nortestosterone sodium sulfate (7.2) (chromatographed as 19nortestosterone) Limit of detection: 64 ng/mL Limit of quantitation: 212 ng/mL
KEY WORDS

horse; pharmacokinetics; SPE

REFERENCE Weidolf,L.O.G.; Chichila,T.M,P.; Henion,J.D. Screening, confirmation and quantification of boldenone sulfate in equine urine after administration of boldenone undecylenate (Equipoise), J. Chromatogr.,

1988, 433, 9-21.

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Boldine
Molecular formula: C19H21NO4 Molecular weight: 327.38 CAS Registry No.: 476-70-0 Merck Index: 1355

SAMPLE

Matrix: blood, urine Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue with 50 |xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of maximum absorbance. This will not necessarily be the optimal wavelength for the separation. Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLC VARIABLES

Guard column: 20 mm long Symmetry C18 Column: 250 X 4.6 5 |xm Symmetry C8 (Waters) Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A: B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial conditions for 7 min. Column temperature: 30 Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5 mL/min) Injection volume: 10-30 Detector: UV 218.1
CHROMATOGRAM Retention time: 8.068 KEY WORDS

whole blood
REFERENCE Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.