Major Metabolites In Human Plasma Research Articles

Assay of TNP-470 and its two major metabolites in human plasma by high-performance liquid chromatography-mass spectrometry.

In this study, a sensitive, specific assay for the determination of TNP-470 and its two major metabolites M-IV (also know as AGM-1883) and M-II in human plasma is reported. The assay involves liquid-liquid extraction of acidified plasma followed by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. A liquid-liquid extraction using an organic solvent mixture (methyl-tert-butyl-ether-hexane, 1:1, v/v) is used in place of solid-phase extraction because it provides consistent recoveries for all analytes, including the internal standard. Retention times for the analytes and internal standard are less than 7 min. Within- and between-day precision is < or = 6.5% and < or = 13.3% relative standard deviation, respectively, for the three analytes. The lower limits of quantitation are 0.25, 0.5, and 1.0 ng/mL for TNP-470, M-IV, and M-II, respectively.

Simultaneous analysis of flunitrazepam and its major metabolites in human plasma by high performance liquid chromatography tandem mass spectrometry

A sensitive and specific high performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (HPLC–APCI–MS–MS) method has been developed for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, in human plasma. After the addition of a deuterium labelled internal standard of flunitrazepam, plasma samples were extracted using Oasis® MCX solid phase extraction cartridges. The compounds were separated on a 5 μm Symmetry C18 (Waters) column (3.0×150 mm, i.d.) with a step gradient of acetonitrile-0.1% formic acid at a flow rate of 0.6 ml/min. The overall extraction efficiency was more than 89% for all three compounds. The limits of detection were 0.25 g/l for flunitrazepam, 0.5 μg/l for 7-aminoflunitrazepam, and 2.0 μg/l for N-desmethylflunitrazepam. Within-run accuracies for quality-control samples were between 92.5 and 101.3% of the target concentration, with coefficients of variation <8%. The proposed method enables the unambiguous identification and quantitation of flunitrazepam and its major metabolites in both clinical and forensic specimens.

Sensitive and specific liquid chromatographic–tandem mass spectrometric assay for dihydroergotamine and its major metabolite in human plasma

A sensitive and specific procedure for the simultaneous determination of dihydroergotamine (DHE) and its 8′-hydroxylated metabolite (8′-OH-DHE) in human plasma was developed and validated. The analytes were extracted from plasma samples by liquid–liquid extraction, separated through a Zorbax C 18 column (50×2.1 mm I.D.) and detected by tandem mass spectrometry with an electrospray ionization interface. Caroverine was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 and 11.0 pg/ml for DHE and 8′-OH-DHE, respectively. The intra- and inter-run precision was measured to be below 9.1% for both DHE and 8′-OH-DHE. The inter-run accuracy was within 4% for the analytes. The overall extraction recoveries of DHE and 8′-OH-DHE were determined to be about 58 and 52% on average, respectively. The chromatographic run time was approximately 2.5 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of DHE and 8′-OH-DHE in a pharmacokinetic study of volunteers who received DHE orally.

The enantioselective determination of chlorpheniramine and its major metabolites in human plasma using chiral chromatography on a β-cyclodextrin chiral stationary phase and mass spectrometric detection

A sensitive enantioselective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of plasma concentrations of (−)( R)- and (+)( S)-chlorpheniramine (CP) and their metabolites, desmethyl-chlorpheniramine (DCP), didesmethyl-chorpheniramine (DDCP) and chlorpheniramine N-oxide (CPNO). Enantioselective separations were achieved on a β-cyclodextrin chiral stationary phase (CYCLOBOND I 2000™) with a mobile phase consisting of diethylamine acetate (0.25%, pH 4.4):methanol:acetonitrile {85:7.5:7.5, (v/v/v)} and a flow-rate of 0.5 ml/min. For CP, the enantioselectivity ( α) of the separation was 1.12 with a resolution factor ( R s) of 1.17. The method was validated for CP by using mass spectroscopy detection (MSD). Concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Extracted calibration curves were linear from 0.13 to 50.00 ng/ml for each enantiomer. The method was applied to samples from two clinical studies.

Determination of lodenosine and its major metabolite in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A sensitive and selective method for the determination of 2'-beta-fluoro-2',3'-dideoxyadenosine (lodenosine, F-ddA), an experimental anti-AIDS drug, and its major metabolite, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), in human plasma was developed and validated. The procedure employs two internal standards and a simple ultrafiltration step followed by chromatography on a Betasil C(18) minibore column. An in-line valve is used to remove salts before reaching the ion source. Detection is by electrospray ionization tandem mass spectrometry with selected reaction monitoring. The method has a limit of quantitation of 4 ng ml(-1) (16 nM) for F-ddA and 8 ng ml(-1) (32 nM) for F-ddI with a linear range up to 2000 ng ml(-1) (7.9 microM) for each. Predicted concentrations from a three-day validation study were within 5% of the nominal values for F-ddA and 16% for F-ddI. Intra- and inter-assay precision, as measured by relative standard deviation, was 13% or better for both compounds. To achieve good reproducibility, many variables related to the electrospray ionization were optimized for both precision and sensitivity. The method was successfully employed to analyze samples and evaluate plasma pharmacokinetics from a Phase I clinical trial.

Liquid−Liquid Extraction in the 96-Well Plate Format with SRM LC/MS Quantitative Determination of Methotrexate and Its Major Metabolite in Human Plasma

A method involving the semirobotic liquid-liquid extraction (LLE) in deep-well 96-well plates was developed for the quantitation of the anti-cancer/antiinflammatory drug methotrexate (MTX) and its major metabolite, 7-hydroxymethotrexate (7OH-MTX) in human plasma. The extraction time for the sample preparation was relatively short with four 96-well plates (384 samples) prepared in approximately 90 min by one person. The sample extracts were each analyzed within 1.2 min using a positive ion turbo-ionspray selected reaction monitoring liquid chromatography/mass spectrometric (SRM LC/MS) method in which 768 samples were easily analyzed within 22 h (maximum of 820 samples in 24 h). Deuterated internal standards, MTX-d3 and 7OH-MTX-d3, were used. The calibration curves for MTX and 7OH-MTX were linear (R2 > 0.997) and ranged from 0.5 to 250 and 0.75 to 100 ng/mL, respectively. The limit of quantitation (LOQ) for MTX and 7OH-MTX was 0.5 and 0.75 ng/mL, respectively; persistent carryover from the autosampler limited the LOQ achievable. The limit of detection (LOD) was 0.05 ng/mL for MTX and 0.1 ng/mL for 7OH-MTX. The intra- and inter-assay precision and accuracy did not exceed 15% for both MTX and 7OH-MTX. The recoveries were 61% for MTX and 47% for 7OH-MTX. The method was validated and demonstrated to be robust with high precision and accuracy.

Simultaneous Determination of Omeprazole and its Metabolites in Human Plasma by HPLC using Solid-phase Extraction

A rapid, simple and sensitive HPLC assay was developed for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 μL) was injected on an μBondapak C18 reversed-phase column (4.6 mm i.d., 250 mm; 10 μm). The mobile phase consisted of 0.05 m phosphate buffer (pH 7-5) and acetonitrile (75:25, v/v) at a flow rate of 0–8 mL min−1. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50–2000 ng mL−1. The minimum detection limits were 10, 10 and 15 ng mL−1 for omeprazole, omeprazole sulphone and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg omeprazole. The extraction procedure and HPLC method is simple, precise and quick, and suitable for the study of pharmacokinetic disposition and metabolism of omeprazole, which is extensively metabolized in man.

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H 3PO 4). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C 6 silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.

Liquid chromatographic determination of irinotecan and three major metabolites in human plasma, urine and feces

A new simple reversed-phase high-performance liquid chromatographic method was developed for the determination of irinotecan (CPT-11) and three metabolites in human plasma, urine and feces homogenate. The metabolites of interest were 7-ethyl-10-hydroxycamptothecin (SN-38), its β-glucuronide derivative (SN-38G) and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (RPR 121056A; also referred to as APC). Sample pretreatment from the various biological matrices involved a rapid protein precipitation with simultaneous solvent extraction of 250-μl aliquots of sample with 500 μl of methanol–5% (w/v) aqueous perchloric acid (1:1, v/v). Separation of the compounds was achieved on an analytical column packed with Hypersil ODS material (100×4.6 mm I.D., 5 μm), and isocratic elution with a mixture of methanol–0.1 M ammonium acetate containing 10 m M tetrabutylammonium sulphate (30:70, v/v), pH 5.3 (hydrochloric acid). The column effluent was monitored at excitation and emission wavelengths of 355 and 515 nm, respectively. Results from a 4-day validation study indicated that this single-run determination allows for simple, simultaneous and rapid quantitation and identification of all analytes with excellent reliability. The described procedure permits the analysis of patient samples, and will be implemented in future studies to investigate the complete metabolic fate and disposition of CPT-11 in cancer patients.

Rapid and simple chromatographic method for the determination of diazepam and its major metabolites in human plasma and urine

A simple, rapid, sensitive and selective HPLC method has been developed for the analysis of diazepam (DZP) and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP) and oxazepam (OZP), in plasma and urine, using clonazepam (CZP) as the internal standard and chloroform as the extracting solvent, with a 10 ng/ml limit of quantitation for the four assayed drugs, and an average (±S.D.) recovery of 87.7±6.46%, 92.9±5.31%, 91.4±4.01% and 91.7±2.68% for DZP, DMDZP, TZP and OZP, respectively (from plasma), and 89.6±2.26%, 90±4.24%, 87.45±0.64% and 94.50±0.71% for DZP, DMDZP, TZP and OZP, respectively (from urine). The method has also proved to be selective and reproducible.

Identification and determination of phase I metabolites of propafenone in rat liver perfusate

Propafenone (PF) is a class 1C antiarrhythmic agent. To study the mechanisms of PF interactions with dietary nutrients in isolated, perfused rat livers, metabolites of PF in liver perfusate were identified and an analytical method was developed for these metabolites plus parent drug. Identification of phase I metabolites was performed using HPLC/MS equipped with a Lichrospher RP-18 column and tandem mass spectrometry (MS/MS) with electrospray and atmospheric pressure chemical ionizations. Three major metabolite peaks, whose protonated molecular ions were m/ z 358, 358 and 300, were identified as a propafenone derivative hydroxylated in the ω-phenyl ring ( ω-OH-PF), 5-hydroxypropafenone (5-OH-PF), and N-despropylpropafenone ( N-des-PF). The levels of ω-OH-PF, 5-OH-PF, N-des-PF and PF were determined simultaneously by HPLC with UV detection at 210 nm and a mobile phase of 0.03% triethylamine and 0.05% phosphoric acid in water-acetonitrile-methanol (45:20:35, v/v/v) after extraction with 5 ml diethyl ether at pH 10.0 and evaporation of solvent under nitrogen. The results revealed that ω-OH-PF, which was not found in humans, was the major metabolite of PF in rat liver perfusate, not 5-OH-PF which is the major metabolite in human plasma.

Mangafodipir trisodium injection, a new contrast medium for magnetic resonance imaging: detection and quantitation of the parent compound MnDPDP and metabolites in human plasma by high performance liquid chromatography

Manganese(II) dipyridoxyl diphosphate (MnDPDP) is the active component of mangafodipir trisodium injection (Teslascan ®), a new contrast medium for magnetic resonance imaging. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of MnDPDP and its five major metabolites in human plasma, i.e. the dephosphorylation products MnDPMP (manganese(II) dipyridoxyl monophosphate) and MnPLED (manganese(II) dipyridoxyl ethylenediamine diacetate) and the corresponding substances obtained after transmetallation with zinc (ZnDPDP, ZnDPMP and ZnPLED). Heparinized blood samples from patients receiving mangafodipir trisodium injection were immediately mixed with solid trisodium phosphate dodecahydrate to obtain pH 10.0 ± 0.2 in order to inhibit further in vitro dephosphorylation and transmetallation. The plasma thus obtained was ultrafiltrated prior to HPLC analysis. The chromatographic separation was obtained using a mixed-bed resin with both anion exchange and reversed-phase functions (OmniPac ™ PAX-500) using isocratic elution and UV detection at 310 nm. With an injection volume of 50 μl, the limit of quantitation (LOQ) values were 0.8–2.3 μM for the Mn compounds and 0.1–0.8 μM for the Zn compounds. The between-run accuracy of spiked plasma samples was in the range 97.5–106.7%, with a precision in the range 3.1–9.0%. The best fit calibration curves were obtained using non-linear regression according to the equation Y = A + BX M in the concentration range from LOQ to 100 μM. Long-term storage of spiked plasma samples for three months at − 20°C demonstrated the required stability with recovery values within 85–115% of MnDPDP and its five metabolites.

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection.

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9 -anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The organic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The eluate was reacted on-line with a mixture of tris(2,2'-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 10 ng/ml, respectively. This method has been used for the determination of 1 in samples from clinical trials.

Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4'-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabase C18 analytical column (5 microns particle size; 250 x 4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantiation were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6-15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C 18 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C 2 (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.

Disposition of CS-670, a novel nonsteroidal anit-inflammatory drug, and its metabolites in healthy human volunteers.

CS-670, a novel nonsteroidal anti-inflammatory drug, is a racemic prodrug. Plasma concentrations and urinary excretion of CS-670 and its metabolites were determined in experimental subjects after oral administration at a single 120 mg dose. CS-670 and four metabolites, the saturated ketone (M-A), unsaturated-alcohol (M-B), cis-alcohol (M-C), and trans-alcohol (M-D), were quantitated by GC-MS. The major metabolites in human plasma were M-B, M-C, and M-D and their terminal half-lives (t1/2) were 0.9, 2.6, and 1.2 h, respectively. The total recovery in the urine was 26% of the dose, but unchanged CS-670 accounted for less than 2% over a 48 h period. In addition, the absolute configurations of the metabolites were examined by HPLC after derivatization with chiral reagents. It was found that the configuration of the propionic acid moiety of the metabolites, M-B, M-C, and M-D, in human plasma, was rapidly inverted from (-)-(R) to the (+)-(S) configuration in stereoselective biotransformation. Furthermore, the configurations of the 1'- and 2'-carbons of M-C and M-D, were found to be (1'R, 2'S) and (1'R, 2'S), respectively. These results show that CS-670 is readily biotransformed by chiral inversion of the 2-arylpropionic acid moiety and stereoselective reduction of the alpha, beta-unsaturated ketone moiety in humans.

Simultaneous determination of granisetron and 7-hydroxygranisetron in human plasma by high-performance liquid chromatography with fluorescence detection.

A highly sensitive high-performance liquid chromatographic method for the determination of granisetron, a novel antiemetic agent for cancer therapy, and its pharmacologically active and major metabolite in human plasma is described. Methylation of the metabolite with trimethylsilyldiazomethane was employed for sample pretreatment. As a result of this treatment, the originally non-fluorescent metabolite could be detected simultaneously with granisetron using fluorescence detection. Both the compounds and the internal standard added were isocratically well resolved from endogenous peaks in plasma on an ODS column. The concentrations of the compounds were proportional to the peak-area ratios over the practical determination ranges of 0.2-100 ng/ml for granisetron and 0.1-50 ng/ml for the metabolite. In tests, reproducibilities for granisetron at 1 ng/ml and the metabolite at 0.5 ng/ml as determined by the relative standard deviation (n = 3) were less than 3.98 and 7.23%, respectively, in a single run.

Simultaneous determination of chloroquine, proguanil and their metabolites in human biological fluids by high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of chloroquine, proguanil and their major metabolites in human plasma, erythrocytes and urine. After a liquid—solid extraction on a Bond Elut® C8 cartridge, the compounds are separated on a C8 Lichrospher 60 RP select B column by isocratic elution; the mobile phase is water—acetonitrile—methanol (78:28:4, v/v/v) with 0.5 M ammonium formate and 0.075 M perchloric acid. The eluent is monitored with an ultraviolet detector at 254 nm. The lower limits of quantification in plasma are near 6.0 ng ml −1 for chloroquine and near 9.0 ng ml −1 for proguanil. No chromatographic interference can be detected from endogenous compounds or from other antimalarial drugs. The method is accurate and precision is good with inter- and intra-assay relative standard deviations lower than 6.8% for plasma samples. N-(2-6 dichlorobenzylidene amino)guanidine is used as an internal standard. The chromatographic procedure takes 35 min and can be used for therapeutic drug monitoring and clinical studies.

High-Performance Liquid Chromatographic Assay with Ultraviolet Detection for the Analysis of BW 1370U87 and Its Two Major Metabolites in Human Plasma

Abstract A sensitive and selective high performance liquid chromatographic assay with UV detection was developed for the analysis of BW 1370U87, a novel MAO-A inhibitor, and its major metabolites, BW 183U88 and BW 1003U88 in human plasma. The compounds were isolated from plasma by liquid extraction with methyl-t-butyl ether. The extracts were chromatographed on a LiChrospher 100 RP-8 column (4.6 mm × 25 cm) using a gradient elution with a mobile phase consisting of acetonitrile-water containing 0.025% phosphoric acid (25:75, v/v, initial conditions increasing to 40:60, v/v, over 25 min). The compounds were detected at 226 nm with a lower limit of quantitation of 10 ng/ml. The accuracy and precision of the assay, expressed as the percent deviation of measured values from true values and the percent coefficient of variation, respectively, were < 8%, at all concentrations. The assay has been successfully applied to the analysis of samples from a clinical pharmacokinetic study in man.