Journal of Pharmaceutical and Biomedical Analysis

30 (2003) 1611 /1619

www.elsevier.com/locate/jpba

Short communication

Simultaneous determination of arylpropionic acidic non-

steroidal anti-inflammatory drugs in pharmaceutical
formulations and human plasma by HPLC with UV detection
Yen Sun, Kumiko Takaba, Hideaki Kido, Mihoko N. Nakashima,
Kenichiro Nakashima

Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-
8521, Japan

Received 8 August 2002; received in revised form 19 September 2002; accepted 20 September 2002

Abstract

A simple and sensitive high-performance liquid chromatography-UV detection method was developed for the
simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) having an arylpropionic acid moiety
in pharmaceutical formulations and human plasma. Isocratic separation was employed on ODS column (250/4.6 mm
i.d., 5 mm) at ambient temperature. The mobile phase consisted of acetonitrile, phosphate buffer (pH 3.5; 50 mM),
methanol and tetrahydrofuran. The NSAIDs in the eluent were monitored under a wavelength-programme to provide
their maximum absorbance. Mefenamic acid was used as an internal standard. Drugs were found to be 96.8 /101.9% of
their label claim in pharmaceutical formulations. One hundred microliters of human plasma samples were pretreated
with a simple liquid-liquid extraction using ethyl acetate. The detection limits of compounds studied at a signal-to-noise
ratio of 3 were 11.5 /75 ng/ml in human plasma samples. The proposed method is simple, selective and could be
applicable for routine analysis of arylpropionic acidic NSAIDs in pharmaceutical as well as in human plasma samples.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Non-steroidal anti-inflammatory drugs; Pharmaceutical formulation; Human plasma; HPLC-UV detection

1. Introduction antipyretics and anti-inflammatory activities and

are widely used in the treatment of acute and
Arylpropionic acid derivatives belong to a chronic pain, osteoarthritis, rheumatoid arthritis
relatively new group of non-steroidal anti-inflam- and related conditions. Diclofenac is also a
matory drugs (NSAIDs), which have analgesics, relatively new NSAID that is being widely pre-
scribed in Japan. The pharmacological actions of
NSAIDs are related to inhibition of cyclooxygen-

Corresponding author. Tel.: /81-95-847-1111x2557; fax:
ase (COX), a key enzyme of prostaglandin bio-
/81-95-842-3549
E-mail address: naka-ken@net.nagasaki-u.ac.jp (K. synthesis at the site of inflammation. The
Nakashima). simultaneous measurement of these NSAID con-
0731-7085/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 7 3 1 - 7 0 8 5 ( 0 2 ) 0 0 5 4 9 - 6
1612 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619

centrations in biological samples is required in of ibuprofen at 254 nm [10]. Generally, although it

clinical and toxicological screening, pharmacoki- is almost impossible that one patient would be
netic studies, as well as in therapeutic monitoring. prescribed for more than one kind of NSAIDs at
Furthermore, it is also very important to precisely the same time, simultaneous determination of
quantify these NSAIDs in pharmaceutical formu- these widely used NSAIDs still has these advan-
lations for quality control Fig. 1. tages: (1) several drugs could be analysed by using
Until now, a variety of chromatographic meth- the same separation condition which might be very
ods have been proposed for the determination of convenient for clinical and pharmaceutical routine
commercially available NSAIDs in pharmaceutical analysis; (2) identification of the peak of NSAIDs
formulations and biological fluids, mainly by gas could be carried out accurately and expeditiously
chromatography (GC) [1 /4], spectrofluorometry by using a simultaneous analytical system; and (3)
[5], HPLC with fluorescence detection [6], ultra- for drug-drug interaction study, it might require a
violet (UV) [7 /16], electrochemical detection simultaneous measurement system to detect more
(ECD) [17], as well as capillary electrophoresis than one drug in biological samples.
(CE) [18]. GC methods are time-consuming and As sample pretreatment, deproteination, liquid /
complex and have been largely replaced by HPLC liquid extraction and solid-phase extraction have
methods. Among HPLC methods, generally they been used. Solid-phase extraction was quite time-
only concentrated on a single or a few compounds. consuming; deproteination is simple, but since the
In other cases, gradient program for separation is sample is diluted, the sensitivity is relatively low.
needed. Hirai et al. reported a simultaneous In this study, considering that simple manipula-
analysis of 12 kinds of NSAIDs in human urine tion and widely used UV detector with no special
[11]. Owen et al. developed a method for determin- assembly might be convenient and suitable for
ing seven kind of NSAIDs simultaneously in routine work, an isocratic separation of NSAIDs
human plasma, yet, under the described condition, having an arylpropionic acid moiety and diclofe-
ketoprofen and naproxen were not sufficiently nac by using reversed phase HPLC and UV
separated and also the assay was not sufficiently detection was developed. The proposed method
sensitive to measure the therapeutic concentration was successfully applied to the determination of

Fig. 1. Chemical structures and abbreviations of NSAIDs studied.

 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619 1613

Fig. 2. Chromatograms of NSAIDs in pharmaceutical formulations. 1: KEP in Menamim; 2: NAP in Naixan; 3: FLB in Floben; 4:
DCL in Voltaren; 5: IBP in Brufen; and 6: MFA (I.S.).

each compound in human plasma sample, as well (NAP) was obtained from ICN Biomedical Co.
as the commercially available pharmaceutical for- (OH). Fenoprofen (FEP) was purchased from
mulations in Japan. Sigma Chemical Co. (Steinheim, Germany). So-
dium hydrogenphosphate, sodium acetate, acetic
acid, phosphoric acid, methanol, acetonitrile and
2. Experimental ethyl acetate were of analytical reagent grade
(Wako). Water was passed through a pure line
2.1. Materials and reagents WL21P (Yamato Sciences, Tokyo, Japan).

Ketoprofen (KEP), flurbiprofen (FLB), ibupro- 2.2. Apparatus

fen (IBP), diclofenac sodium (DIC), mefenamic
acid (MFA) were purchased from Wako Pure The HPLC system consisted of a Jasco 880-pu
Chemical Industries (Osaka, Japan). Naproxen pump (Tokyo, Japan), a Jasco 875-UV multi-
1614 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619

wavelength UV/VIS detector, a Rheodyne 7125 the mobile phase was added to make the volume
sample injector with a 20-ml loop (Cotati, CA) and up to 1.0 ml. An aliquot (20 ml) of each solution
a Rikadenki R-01 recorder (Tokyo, Japan). Chro- was injected onto the column.
matographic separations were carried out on a
Daisopak ODS column (SP-120-5-ODS-BP, 2.6. Determination of NSAIDs in human plasma
250 /4.6 mm i.d., Daiso, Osaka, Japan).
Human blood samples were collected from
2.3. Preparation of standard solutions healthy volunteers in our laboratory. The blood
was drawn into the test tube including EDTA.
Standard stock solutions of KEP, NAP, FLB, After centrifugation at 1000 g for 10 min, the
IBU, DIC (10 mg/ml), FEP (1.0 mg/ml) and MFA plasma was taken and kept in a freezer (/20 8C)
(2.0 mg/ml) as an internal standard (I.S.) were until analysis.
prepared by dissolving appropriate amounts of For drug analysis, 100 ml of human plasma were
them in methanol. These stock solutions were transferred to a test tube and 200 ml of phosphate
subsequently used in the preparation of working buffer (pH 2.5; 50 mM) was added and mixed well.
standards by further dilution with methanol. All To the mixture, 700 ml of ethyl acetate was added
stock solutions were kept in refrigerator at 4 8C, and vortex-mixed for 1 min. After centrifugation
which were stable for at least 3 months. NSAIDs at 1000 /g for 10 min, the organic layer (600 ml)
spiked plasma samples were prepared as follows: was transferred to a test tube. The extraction
after evaporating a proper volume of each working procedure was repeated once and the collected
standards, 100 ml of drug-free plasma were added ethyl acetate layer was evaporated to dryness.
to the residue to obtain the final concentration Thereafter, 200 ml of mobile phase were added to
range of 0.1 /100 mg/ml. the residue, centrifuged at 1000 g for 10 min and
20 ml of the supernatant was injected onto the
2.4. Chromatographic conditions HPLC system.

The NSAIDs were isocratically separated with

acetonitrile-acetate buffer (pH 3.5; 0.1 M)-metha- 3. Results and discussion
nol (35:40:25, v/v/v) as an eluent at a flow rate of
1.0 ml/min. The effluent was monitored at 255 nm 3.1. Chromatographic separations
for 0 /11.6 min, 240 nm for 11.6 /16.8 min and 220
nm for 16.8 /35 min. The chart speed was set at 2.5 In this study, an isocratic separation of NSAIDs
mm/min. in standard solution was carried out by using a
mixture of acetonitrile-acetate buffer (pH 3.5; 0.1
2.5. Determination of NSAIDs in pharmaceutical M)-methanol (35:40:25, v/v/v). The capacity factor
formulations (k ?) value of each compound remarkably de-
creased in the pH value of mobile phase above
The contents of ten capsules of KEP and ten 4.5, whereas there were little changes up to pH 4.0;
tablets of NAP, FLB, DIC and IBP were finely the pH 3.5 was chosen as the optimal condition.
ground. An accurately weighed powdered sample The proposed separation condition was applied to
containing the labeled amount of each drug was the analysis of NSAIDs in pharmaceutical for-
transferred to a 100-ml volumetric flask. The mulations (Fig. 2). However, FEP and DIC were
volume was adjusted with methanol and the interfered with the endogenous peak from plasma
resultant solution was sonicated for 5 min. A by using acetate buffer in mobile phase. After
portion of the solution was then filtered through a changing to a phosphate buffer (pH 3.5; 50 mM)
0.45 mm filter (Kanto Chemical Co., Tokyo, and the slight modification of acetonitrile
Japan). To each 100 ml of filtrate, 100 ml of the and methanol composition in mobile phase, a
I.S. solution (0.5 mg/ml of MFA) were added and complete separation of all compounds in plasma
 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619 1615

Fig. 3. Chromatograms of NSAIDs in human plasma. (A) Drug-free human plasma; (B) human plasma spiked with each drug at 1.0
mg/ml. 1: KEP; 2: NAP; 3: FEP; 4: FLB; 5: DCL; 6: IBP; and 7: MFA (I.S.).

sample could be achieved at acetonitrile-phosphate 3.2. Selection of detection wavelength

buffer (pH 3.5; 50 mM)-methanol-tetrahydrofuran
(25:40:35:1.5, v/v/v) (Fig. 3). As shown in Table 1, each compound exhibited
different maximum UV absorbance. In order to
detect NSAIDs sensitively, a wavelength-pro-
gramme was performed in this study. For human
plasma analysis, the program was set as follows:
255 nm (0 /13.6 min), 240 nm (13.6 /20.6 min) and
Table 1 220 nm (20.6 /42.0 min). For pharmaceutical
Maximum UV absorption of NSAIDs formulation analysis, each drug was monitored
Compound lmax (nm)a at its maximum wavelength. Under these condi-
tions, standard calibration curves showed good
KEP 256 linearity (r
/0.999), ranging from 0.05 to 100 mg/
NAP 232
ml for KEP, NAP, FEP; 0.1 /100 mg/ml for FLB,
FEP 210
FLB 246 IBP, DIC.
DIC 274
IBP 226 3.3. Determination of NSAIDs in pharmaceutical
MFA 222 formulations
a
UV absorption of each compound was measured in mobile
phase [acetonitrile/acetate buffer (pH 3.5; 0.1 M)/ The accuracy of the proposed method was
methanol/35:40:25, v/v/v]. evaluated by recovery assays. In this study, the
1616 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619

recovery assessment was made on the formulation 3.4. Determination of NSAIDs in human plasma
samples instead of preparing placebos. Thus,
known amounts of each compound were spiked 3.4.1. Linearity
into their corresponding formulation at two levels The calibration curves were prepared over the
and each spiked matrices were prepared in four concentration range of 0.1 /100 mg/ml in human
replicates. The recovery was calculated as follows: plasma for each compound by assaying in dupli-
cates at eight different concentrations. A linear
relationship was obtained between the peak height
Recovery (%) ratio and concentration for each drug in spiked
[(measured conc: human plasma samples. As shown in Table 4, the
original conc:)=spiked conc:]100 (1) calibration curves were linear over the spiked
range for each compound with the good correla-
tion coefficient (r
/0.999). Limit of detections
The precision of the proposed method was (LODs) of each compound were 11.5 /75 ng/ml at
performed by spiking each compound to the a signal-to-noise ratio of 3. Compared with those
selected formulation on different days. The preci- of other publications [10,11], the proposed method
sion (% R.S.D.) for all the studied components in was three to 30 times more sensitive.
different dosage forms were B/6.7 and 4.9% for
intra-day and inter-day assay, respectively (Table 3.4.2. Extraction recovery
2). Recovery of the extraction procedure was de-
The method was applied to seven different termined for each compound at 1.0 mg/ml. The
pharmaceutical formulations (capsules and ta- recovery of I.S. was determined at 2.0 mg/ml as its
blets) for determining the content of NSAIDs. working concentration. Recoveries were calculated
As shown in Table 3, the value ranged of 96.8 / by comparing the peak heights of each compound
101.9% as the percentage of the label claim. spiked in plasma, to those of standards in the

Table 2
Recoveries and precision of the proposed method in pharmaceutical formulations

Compound Spiked level Conc. Found Recovery Precision

(mg/ml) (mg/ml) (Accuracy, %) (n /4)
Intra-day (n/4) (R.S.D.%) Inter-day (n/4) (R.S.D.%)

KEP 0 0.169/0.023 / 1.1 0.5

0.07 0.239/0.028 100.2 4.9 4.9
0.14 0.309/0.023 99.9 1.2 2.8
NAP 0 0.089/0.002 / 2.4 3.6
0.04 0.129/0.003 99.9 6.7 3.6
0.16 0.249/0.001 100.1 0.3 2.8
FLB 0 0.0489/0.002 / 1.4 1.5
0.025 0.0719/0.001 95.2 4.8 3.8
0.075 0.1239/0.001 100.5 0.2 2.8
IBP 0 0.099/0.005 / 0.6 3.6
0.045 0.139/0.005 98.1 1.4 4.8
0.09 0.189/0.004 100.5 0.4 3.7
DIC 0 0.0189/0.0001 / 1.3 2.0
0.01 0.0289/0.0005 89.4 1.8 2.9
0.04 0.0579/0.0002 101.2 0.5 1.2
 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619 1617

Table 3
Contents of NSAIDs in pharmaceutical formulations and OTC drugs

Drug Dosage form Claimed Founda Contentb

(mg) (mg) (%)

Pharmaceutical formulations
Menamim (KEP) Capsule 150 151.59/4.55 101.0
Naixan (NAP) Tablet 100 101.29/1.09 101.2
Froben (FLB) Tablet 40 38.79/1.17 96.8
Voltaren (DIC) Tablet 25 24.99/1.16 99.6
Brufen (IBP) Tablet 100 101.99/2.75 101.9
OTC drugs
EVE A (IBP) Tablet 75 73.49/0.24 97.8
NARON ACE (IBP) Tablet 72 69.79/1.90 96.8
a
Mean9/S.D. of four replications.
b
Content (%)/Found/Claimed/100.

mobile phase. Several different solvents (dichlor- replicate samples at each concentration were
omethane, hexane, chloroform, ethyl acetate) were analyzed for the intra-assay assessment. For the
studied for liquid-liquid extraction and ethyl inter-day precision, analysis of the spiked plasma
acetate was chosen as adequate. Recoveries of at the same concentrations of each drug was
each compound were also tested at different pH by performed on 5 different days. Precision and
acidified with 50 mM phosphate buffer (pH 1.5 / accuracy are shown as R.S.D.% and percent
5.0). The maximum recovery of each drug was accuracy [(mean observed conc./spiked conc.) /
obtained at pH 2.5. As shown in Table 5, 100], respectively. The results are given in Table
recoveries for NSAIDs were 97.1 /114.6% at 5. The intra- and inter-day precision of the assay
studied concentrations, which were relatively were observed to be B/8.4 and 10.1%, respectively.
higher than those of 73.1 /95.1% in other pub- The assay accuracy was found to be 90.0 /111.5%
lished method [11]. for all compounds.

3.4.3. Assay accuracy and precision 3.4.4. Stability

Assessment of the intra-day accuracy and pre- The freeze-thaw stability of NSAIDs in plasma
cision of the method were performed in several was assessed over two freeze-thaw cycles. Each
different lots of drug-free plasma samples spiked compound was spiked in plasma at concentration
with each drug at low (0.1 mg/ml), middle (1.0 mg/ of 1.0 mg/ml, which was frozen at /20 8C and
ml) and high (10 mg/ml) concentrations. Six thawed at room temperature for two consecutive

Table 4
Calibration curves and limit of detections (LODs) of NSAIDs in human plasma

Compound Concentration range Equation r LOD (S/N/3)

(mg/ml) (ng/ml)

KEP 0.1 /100 Y/1.86X/0.25 0.999 11.5

NAP 0.1 /100 Y/0.46X/0.14 0.999 75.0
FEP 0.1 /100 Y/0.49X/0.046 0.999 75.0
FLB 0.1 /100 Y/1.08X/0.13 0.999 13.6
DIC 0.1 /100 Y/0.95X/0.22 0.999 30.0
IBP 0.1 /100 Y/0.51X/0.0034 0.999 42.8
1618 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619

Table 5
Intra-, inter-day accuracy, precision and recoveries of NSAIDs in human plasma

Compound Nominal Found conc. Accuracy R.S.D. (%) Recovery

(mg/ml) (mg/ml) (%) (%)
Intra-day (n/6) Inter-day (n/5)

KEP 0.1 0.119/0.01 111.5 5.0 8.6 97.1

1.0 1.059/0.06 105.4 5.8 4.3
10.0 10.49/0.45 104.4 4.3 3.6
NAP 0.1 0.909/0.01 90.0 5.6 8.8 98.6
1.0 1.029/0.05 102.3 5.0 7.9
10.0 10.79/0.50 107.1 4.7 3.2
FEP 0.1 0.099/0.01 90.1 5.6 8.8 110.0
1.0 0.909/0.02 90.0 2.7 2.6
10.0 10.79/0.59 106.6 5.5 1.6
FLB 0.1 0.109/0.01 103.0 6.7 6.7 97.1
1.0 1.009/0.04 99.8 4.3 4.8
10.0 10.89/0.50 108.8 4.6 3.2
DIC 0.1 0.119/0.01 109.4 8.4 4.7 108.8
1.0 0.999/0.02 99.4 2.5 2.2
10.0 10.39/0.42 103.1 4.1 0.7
IBP 0.1 0.119/0.003 110.7 5.4 10.1 114.6
1.0 1.049/0.03 104.0 3.2 1.9
10.0 10.39/0.46 102.5 4.4 2.3
MFA (I.S.) 2.0 / / / / 100.0

Table 6
Stability of NSAIDs in human plasma

Compound Conc. spiked Conc. measured

(mg/ml) (mg/ml)

Initial Free-thaw stability Stored at room temperature

Cycle 1 Cycle 2 24 h 48 h

KEP 1.0 0.97 0.96 0.99 0.96 1.03

NAP 1.0 1.01 0.96 0.99 0.99 1.07
FEP 1.0 0.98 0.95 1.04 0.96 1.01
FLB 1.0 0.97 0.96 1.01 1.00 1.05
DIC 1.0 1.02 1.00 0.99 0.97 1.03
IBP 1.0 1.00 0.98 1.02 0.99 1.08

times. As shown in Table 6, freeze-thaw stabilities 4. Conclusions

of NSAIDs were evaluated to be 0.99 /1.04 mg/ml,
room temperature stabilities until 48 h were within A simple and sensitive HPLC-UV detection
0.96 /1.08 mg/ml, with spiked concentration of 1.0 method for the simultaneous determination of
mg/ml. Overall results indicate that NSAIDs stu- NSAIDs having arylpropionic acid moiety, as
died are stable in plasma samples following two well as diclofenac was developed. The proposed
freeze-thaw cycles and room temperature storage. method was successfully applied to the determina-
 Y. Sun et al. / J. Pharm. Biomed. Anal. 30 (2003) 1611 /1619 1619

tion of these NSAIDs in pharmaceutical formula- after solid-phase extraction, J. Chromatogr. B 763 (2001)
195 /200.
tions, as well as in the human plasma samples. The
[8] E. Mikami, T. Goto, T. Ohno, H. Matsumoto, K. Inagaki,
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