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    Pravastatin

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    Papaindo
    The document describes sample preparation and chromatography methods for analyzing pravastatin, a drug used to lower cholesterol, in various matrices like blood, feces, urine, and solutions. Sample preparation involves steps like protein precipitation, solid phase extraction, and centrifugation followed by HPLC with detection using UV, fluorescence, or radioactivity. Chromatography conditions include the use of C18 columns with mobile phases containing organic solvents and buffers at varying compositions and pH. Retention times are reported between 3-33 minutes depending on the specific conditions and matrix.

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    Pravastatin

    Uploaded by

    Papaindo
    77 views3 pages
    The document describes sample preparation and chromatography methods for analyzing pravastatin, a drug used to lower cholesterol, in various matrices like blood, feces, urine, and solutions. Sample preparation involves steps like protein precipitation, solid phase extraction, and centrifugation followed by HPLC with detection using UV, fluorescence, or radioactivity. Chromatography conditions include the use of C18 columns with mobile phases containing organic solvents and buffers at varying compositions and pH. Retention times are reported between 3-33 minutes depending on the specific conditions and matrix.

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    By HPLC

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    The document describes sample preparation and chromatography methods for analyzing pravastatin, a drug used to lower cholesterol, in various matrices like blood, feces, urine, and solutions. Sample preparation involves steps like protein precipitation, solid phase extraction, and centrifugation followed by HPLC with detection using UV, fluorescence, or radioactivity. Chromatography conditions include the use of C18 columns with mobile phases containing organic solvents and buffers at varying compositions and pH. Retention times are reported between 3-33 minutes depending on the specific conditions and matrix.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    77 views3 pages

    Pravastatin

    Uploaded by

    Papaindo
    The document describes sample preparation and chromatography methods for analyzing pravastatin, a drug used to lower cholesterol, in various matrices like blood, feces, urine, and solutions. Sample preparation involves steps like protein precipitation, solid phase extraction, and centrifugation followed by HPLC with detection using UV, fluorescence, or radioactivity. Chromatography conditions include the use of C18 columns with mobile phases containing organic solvents and buffers at varying compositions and pH. Retention times are reported between 3-33 minutes depending on the specific conditions and matrix.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
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    The document discusses several methods for extracting, analyzing, and detecting pravastatin and its metabolites from various sample matrices like plasma, feces, urine, and solutions using techniques like HPLC, SPE, and radiolabeling.

    Pravastatin can be extracted from plasma samples using an immobilized antibody column or SPE cartridge. The column or cartridge is conditioned and plasma is added, washed, and the pravastatin is then eluted off.

    Pravastatin and its metabolites can be analyzed using techniques like HPLC with UV or fluorescence detection after extraction from samples. Radiolabeling of starting materials can also be used to extract and detect metabolites. Semi-preparative HPLC can also be used to separate and collect metabolite fractions.

    Pravastatin

    Molecular formula: C23H36O7


    Molecular weight: 423.5
    CAS Registry No.: 81131-70-6 (pravastatin sodium),
    81093-37-0 (pravastatin)

    SAMPLE
    Matrix: blood
    Sample preparation: Condition an immobilized antibody column (preparation details in
    paper) with 4 mL water, 4 mL MeOH: water 80:20, and 4 mL PBS. Add 1 mL plasma to
    the column, let stand for 15 min, wash with two 4 mL portions of water, wash with 4 mL
    PBS, wash with two 4 mL portions of water, elute with 4 mL MeOH. Add 100 (xL 100
    ng/mL IS in water to the eluate, evaporate to dryness, reconstitute with 100 (xL DMF,
    add 100 |xL 47.6 jxg/mL triethylamine in dioxane (Caution! Dioxane is a carcinogen!), add
    100 |xL 51.8 |xL/mL diethyl phosphorocyanidate in dioxane, add 100 |xL 100 jxg/mL Ndansylethylenediamine (Molecular Probes, Inc., Eugene OR) in dioxane, stir for 10 s, let
    stand at room temperature for 10 min, inject a 10 |xL aliquot onto column A and elute to
    waste with mobile phase A. After 7.5 min elute the contents of column A onto column B
    with mobile phase B. After 2.5 min remove column A from the circuit. Elute column B
    with mobile phase B and monitor the effluent from column B.
    HPLCVARIABLES
    Column: A 150 X 4.6 Ultron 300-C4 (Shinwa Chemical Industries); B 150 X 6 Cosmosil
    5C18-AR (Nacalai Tesque)
    Mobile phase: A MeCN: 10 mM pH 2.4 citric acid 30:70; B MeCN: 5 mM pH 2.6 citric acid
    50:50

    Flow rate: 1
    Injection volume: 10
    Detector: F ex 325 (40 mW He-Cd laser); F ex 350 em 530; UV 239
    CHROMATOGRAM
    Retention time: 17.3
    Internal standard: R-416 (Sankyo) (19)
    Limit of quantitation: 0.1 ng/mL (laser fluorescence)
    KEYWORDS
    column-switching; dog; rat; laser; plasma; SPE; derivatization; pharmacokinetics
    REFERENCE
    Dumousseaux, C; Muramatsu, S.; Takasaki, W.; Takahagi, H. Highly sensitive and specific determination of pravastatin sodium in plasma by high-performance liquid chromatography with laser-induced fluorescence detection after immobilized antibody extraction. J.Pharm.Sci., 1994, 83, 16301636

    SAMPLE
    Matrix: blood
    Sample preparation: Condition a 1 mL C2 Bond-Elut SPE cartridge with 2 mL water, 2
    mL MeOH, and 2 mL water. 1 mL Plasma + 1 mL 100 mM pH 7.2 KH2PO4, add to the
    SPE cartridge, wash with 2 mL water, elute with 500 JJLL MeCN-.water 75:25. Evaporate
    the eluate to dryness under reduced pressure, reconstitute with 80 JULL IS solution, cen-

    trifuge, inject a 50 |xL aliquot. (Prepare the IS solution by mixing 2 mL 100 |xg/mL simvastatin p-hydroxyacid in MeOH, 1 mL MeOH, and 1 mL 100 mM pH 5 KH2PO4.)
    HPLC VARIABLES
    Guard column: 20 mm long Supelguard LC-18
    Column: 50 X 4.6 3 jim Supelcosil LC-18
    Mobile phase: MeCN: 50 mM pH 3.5 ammonium phosphate 26:74
    Column temperature: 50
    Flow rate: 1.6
    Injection volume: 50
    Detector: UV 238
    CHROMATOGRAM
    Retention time: 3.0
    Internal standard: simvastatin p-hydroxyacid (4.8)
    Limit of detection: 2 ng/mL
    KEYWORDS
    pharmacokinetics; plasma; SPE
    REFERENCE
    Iacona, L; Regazzi, M.B.; Buggia, I.; Villani, P.; Fiorito, V.; Molinaro, M.; Guarnone, E. High-performance
    liquid chromatography determination of pravastatin in plasma. Ther.Drug Monit., 1994, 16, 191
    195

    SAMPLE
    Matrix: blood, feces, urine
    Sample preparation: Plasma. 2 mL Plasma + 4 mL MeCN, centrifuge at 700 g for 10
    min, remove the supernatant and wash the precipitate twice with 2 mL MeCN: water 2:
    1. Combine the supernatants and evaporate them to dryness under vacuum, reconstitute
    the residue in 1 mL MeCN: water 2:1, centrifuge at 10000 g, remove the supernatant and
    add it to 500 |xL water, centrifuge at 10000 g, inject a 1 mL aliquot. Urine. Centrifuge at
    10000 g, inject an aliquot. Feces. 1 g Homogenized feces + 2 mL MeCN, sonicate for 5
    min, shake in a wrist action shaker for 20 min, centrifuge at 700 g for 10 min. Remover
    the supernatant and wash the precipitate twice with 1 mL MeCN: water 2:1, combine
    the supernatants, inject a 500 |xL aliquot.
    HPLC VARIABLES
    Guard column: present but not specified
    Column: 500 X 9.4 Partisil 10 ODS-3 C18
    Mobile phase: Gradient. MeCN: 10 mM pH 7.2 potassium phosphate buffer containing 5
    mM tetrabutylammonium hydrogen sulphate at 25:75 for 20 min, then to 50:50 over 45
    min, hold at 50:50 for 10 min.
    Flow rate: 4
    Injection volume: 500-1000
    Detector: Collect fractions and measure radioactivity; UV 245
    CHROMATOGRAM
    Retention time: 33
    OTHER SUBSTANCES
    Extracted: metabolites
    KEYWORDS
    plasma; semi-preparative; radiolabeled starting material

    REFERENCE
    Everett, D.W.; Chando, T.J.; Didonato, G.C.; Singhvi, S.M.; Pan, H.Y.; Weinstein, S.H. Biotransformation
    of pravastatin sodium in humans. Drug Metab.Dispos., 1991, 19, 740-748

    SAMPLE

    Matrix: solutions
    Sample preparation: Inject an aliquot of an aqueous solution.
    HPLCVARIABLES

    Column: Cosmosil 5C18-AR (Nacalai Tesque)


    Mobile phase: A MeCN: 10 mM pH 2.4 citric acid 30:70
    Flow rate: 1
    Detector: UV 235
    REFERENCE
    Dumousseaux, C; Muramatsu, S.; Takasaki, W.; Takahagi, H. Highly sensitive and specific determination of pravastatin sodium in plasma by high-performance liquid chromatography with laser-induced fluorescence detection after immobilized antibody extraction. J.Pharm.ScL, 1994, 83, 16301636

    SAMPLE

    Matrix: solutions
    Sample preparation: Centrifuge at 2000 rpm, inject an aliquot.
    HPLCVARIABLES

    Column: 300 X 3.9 jxBondapak


    Mobile phase: MeOH: water: triethylamine: glacial acetic acid 500:500:1:1
    Column temperature: 30
    Flow rate: 1.3
    Detector: UV 238
    CHROMATOGRAM

    Retention time: 17.5 (10.1 hydroxy acid form)


    Limit of detection: 10 ng/mL
    Limit of quantitation: 25 ng/mL
    REFERENCE
    Serajuddin, A.T.; Ranadive, S.A.; Mahoney, E.M. Relative lipophilicities, solubilities, and structurepharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin. J.Pharm.Sci., 1991, 80, 830-834

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