Resolution of enantiomers of the antiarrhythmic drug encainide and its major metabolites by chiral derivatization and high-performance liquid chromatography

Commercially available chiral columns were unable to provide adequate resolution of enantiomers of the antiarrhythmic drug encainide or its major metabolites. The homochiral derivatizing agent, (-)-menthyl chloroformate, was found to react at the tertiary piperidine nitrogen of racemic encainide providing two menthyl carbamate diastereomers. The individual diastereomers could be separated with baseline resolution on normal-phase high-performance liquid chromatography on a silica column. Structures of the derivatives were confirmed by electron impact mass spectrometry and 1H NMR spectroscopy. The method was adapted for the chiral analysis of the major metabolites of encainide. The limit of sensitivity for racemic encainide was 10 ng on column and it was possible to detect a mixture containing (+)- and (-)-encainide in a ratio of 1:99. Preliminary studies indicated that (-)-encainide was O-demethylated to a greater extent than the (+)-enantiomer by rat liver microsomes.     

Analysis of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and Its Phase I and Phase II Metabolites in Mouse Urine Using LC�CUV�CMS�CMS

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is an abundantly present heterocyclic aromatic amine which is found to be carcinogenic in rodents, mice and rats. The biotransformation of PhIP is extensive and involves both the formation of bioactivated as well as detoxification metabolites. In order to understand its carcinogenicity, the metabolism of PhIP needs to be studied. Numerous metabolites of PhIP have been described but, so far, assays for their quantitative determination in biological matrices are scarce. We present the development and application of an assay, using reversed phase liquid chromatography coupled to ultraviolet and mass spectrometry detectors for the quantification of PhIP, three phase I and nine phase II metabolites in urine. Additionally, the identification of two PhIP-sulfates by the use of NMR is presented. Sample pretreatment consisted of straightforward dilution of urine. PhIP and its metabolites were shown to be stable in diluted urine for at least 22 h when stored at 2?C8 °C. Precision of the analysis was within 15%. The assay has been successfully applied for the quantification of PhIP and 12 of its metabolites in urine from mice that received 200 mg kg^?1 PhIP via oral gavage.     

Screening for testosterone, methyltestosterone, 19-nortestosterone residues and their metabolites in bovine urine with enzyme-linked immunosorbent assay (ELISA)

This work reports on the development of rapid enzyme-linked immunosorbent assay for testosterone, methyltestosterone, 19-nortestosterone and their metabolites in real samples of bovine urine. The assays were based on the competition between an immobilised testosterone-BSA conjugate and the analyte for corresponding antibodies, followed by the use of secondary anti-species (α-IgG-HRP) to determine the degree of competition. Urine samples were analysed after 10-fold dilution with the buffer, omitting extraction and hydrolysis. The limits of detection calculated for the original sample were ca. 74, 266 and 131 pg ml⁻¹ (ppt) for testosterone, methyltestosterone and 19-nortestosterone, respectively. In particular, testosterone and methyltestosterone assays offer the advantage to pick up both parent compounds and their major metabolites due to the high cross-reactivity pattern with corresponding antibody used in each assay. The concentration in bovine urine detected by developed method does indicate 19-nortestosterone administration to heifers. The developed assays were applied to urine samples of heifers treated with above androgens.     

Simultaneous determination of cytosine arabinoside, its nucleotides and metabolites by ion-pair high-performance liquid chromatography

Currently available high-performance liquid chromatographic assays for cytosine arabinoside (ara-C) and its metabolites suffer from two major shortcomings: inability to resolve both ara-C and its nucleotides in a single chromatographic step and/or inadequate sensitivity to allow quantitation of intracellular cytosine arabinofuranoside-5'-triphosphate (ara-CTP) without the use of radiolabelled drug. In this paper, we describe a new ion-pairing high-performance liquid chromatographic assay for ara-C in biological samples that can separate ara-C from its nucleotides, metabolites, and naturally occurring ribonucleotides in a single chromatographic step with a lower limit of quantitation of 5 pmol for ara-C and 10 pmol for ara-CTP. Examples of the utility of this assay are shown in studies of intracellular pharmacokinetics of ara-C in cultured human breast cancer cells and in analysis of plasma nucleoside levels in patients receiving high-dose thymidine chemotherapy. We conclude that this assay provides a rapid and versatile system that can be applied to the study of both cellular and plasma nucleoside pharmacokinetics.     

Analysis of the O-methylated metabolites of isoprenaline, adrenaline and noradrenaline in physiological salt solutions by high-performance liquid chromatography with electrochemical detection

This study provides the first report of a sensitive, simple and rapid high-performance liquid chromatographic (HPLC) assay for the simultaneous analysis of isoprenaline and its metabolite, 3-O-methylisoprenaline, in samples of physiological salt solutions. The assay does not require time-consuming sample clean-up or extraction procedures and uses a Nova-Pak C18 column, an isocratic mobile phase and an amperometric detector. In addition, small modifications to the composition of the mobile phase have also provided sensitive assays for noradrenaline and adrenaline and their O-methylated or O-methylated deaminated metabolites (normetanephrine, metanephrine, 3-methoxy-4-hydroxyphenylethylene glycol and 3-methoxy-4-hydroxymandelic acid). These HPLC assays are sufficiently sensitive and rapid to replace the use of [3H]amines and column chromatographic separation of the metabolites for most in vitro studies on the uptake and subsequent metabolism of catecholamines by monoamine oxidase and/or catechol-O-methyltransferase in tissues.     

Determination of Cotinine, 3′-Hydroxycotinine,and Their Glucuronides in Urine by Ultra-high Performance Liquid Chromatography

The measurement of the primary nicotine metabolites, cotinine and trans-3′-hydroxycotinine, is a useful biomarker of nicotine exposure and metabolism genetics for smoking cessation research. Herein is described an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of these primary nicotine metabolites in urine. Urine samples were diluted one hundred-fold with water and introduced into an ultra-high performance liquid chromatography triple quadrupole mass spectrometer using positive ion electrospray ionization with multiple reaction monitoring. Levels of urinary nicotine metabolites: cotinine, trans-3′-hydroxycotinine, and their respective glucuronides were determined directly using deuterated internal standards and compared with indirect determination by enzymatic hydrolysis. The assay was applied to a community sample of smokers’ urine (n = 280). The assay demonstrated satisfactory performance (relative standard deviation of 1.6–6.5 percent at the 1000 nanograms per milliliter level and >98 percent recovery) suitable for application to smoking studies with a run time less than five minutes. The mean (min-max) levels of cotinine and cotinine-glucuronide were 968 (31-3831) and 976 (9-5607) nanograms per milliliter. The mean (min-max) levels of trans-3′-hydroxycotinine and trans-3′-hydroxycotinine-glucuronide were 3529 (13-21337) and 722 (0-4633) nanograms per milliliter. Direct determination of glucuronide metabolites was superior to indirect measurement using enzymatic hydrolysis, where there was evidence of loss of metabolites during sample preparation. A sensitive and selective ultra-high performance liquid chromatography–tandem mass spectrometry assay was successfully developed for the determination of cotinine, trans-3′-hydroxycotinine, and their glucuronides in urine. The rapid and simple sample preparation makes this assay suitable for high throughput studies involving nicotine metabolism phenotype for both cytochrome P450 2A6 and uridine 5′-diphospho-glucuronosyltransferase, smoking prevalence, and cessation studies.     

High-performance liquid chromatographic quantitation of cyclooxygenase and lipoxygenase metabolites of arachidonic acid from rat polymorphonuclear leukocytes

10-Ethyl-10-deaza-aminopterin (10-EdAM) is a novel folic acid antimetabolite currently being tested in phase II clinical trials. We have developed an isocratic high-performance liquid chromatographic method for the quantification of 10-EdAM and metabolites in plasma. Solid-phase extraction was used for sample clean-up. Adequate accuracy was obtained without the use of an internal standard. Fluorometric detection with excitation at 243 nm and emission at 488 nm was used for accurate quantification of samples containing small amounts of drug or metabolites (2.0-4.0 nM, depending on the compound). Ultraviolet detection at 350 nm was only applicable for the analysis of plasma concentrations of 10-EdAM exceeding 50 nM. The usefulness of the assay was demonstrated by the results obtained in a pharmacokinetic study. The assay could separate the parent compound from seven identified and two unknown products.     

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract

A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl.

The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.     

High-performance liquid chromatographic separation of the enantiomers of hydroxychloroquine and its major metabolites in biological fluids using an alpha 1-acid glycoprotein stationary phase.

An enantioselective two-stage off-line assay has been developed for the analysis of hydroxychloroquine and its three major metabolites in biological fluids. The first non-stereoselective stage of the assay (PRP-1 column) separates and quantitates parent drug and metabolites. Fractions containing hydroxychloroquine and each of the metabolites are collected manually, evaporated, reconstituted in mobile phase and re-injected onto an alpha 1-acid glycoprotein column to separate and determine proportions of individual enantiomers. Preliminary results from patients samples indicate that the disposition of hydroxychloroquine and its major metabolites is enantioselective. p6     

Quantitation of Toremifene and its Major Metabolites in Human Plasma by High-Performance Liquid Chromatography Following Fluorescent Activation

Abstract

A highly sensitive reverse-phase high-performance liquid chromatography assay utilizing fluorescence activation has been developed for the quantitative analysis of the anti-estrogenic compound toremifene and its major metabolites, 4-hydroxy-toremifene and N-desmethyl-toremifene. Plasma samples containing various quantities of toremifene and its metabolites were spiked with an internal standard (nafoxidine), extracted with 2% n-butanol in hexane, and irradiated with high intensity ultraviolet light (254 nm). Aliquots of the extracted plasma components were then injected onto a C-18 reversed phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of toremifene, its metabolites, and internal standard was measured at an excitation wavelength of 266 nm. -The sensitivity of this assay was 8.0, 15.0 and 5.0 ng/mL for toremifene, N-desmethyl-toremifene and 4-hydroxy-toremifene, respectively. Linearity was achieved for the concentration range of 25 to 400 ng/mL for all the compounds, with correlation coefficients of greater than 0.994. The assay presented is highly specific, very sensitive and demonstrates reproducible linearity throughout a wide range of clinically relevant plasma toremifene concentrations.     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for quantification of nevirapine and its five metabolites and their pharmacokinetics in baboons

A highly sensitive and specific LC‐MS/MS assay was developed and validated to quantify nevirapine (NVP) and its five metabolites [2‐, 3‐, 8‐, 12‐hydroxyl NVP (OHNVP) and 4‐carboxyl NVP (CANVP)] simultaneously in baboon serum and the assay was used to characterize their pharmacokinetic studies of an oral‐dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolites was 1.0 ng/mL and for 4‐CANVP was 5.0 ng/mL. The between‐run and within‐run precisions and accuracies at four quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboon serum with less than 14% variation and 93–114% accuracies (n = 6), except for the LLOQ for 2‐OHNVP, which had an accuracy of 115.8% for between‐run validation. The pharmacokinetics of NVP and its five metabolites in non‐pregnant baboons by a single‐dose escalation study were also profiled. The major metabolites detected were 4‐CANVP and 12‐OHNVP. 3‐OHNVP and 2‐OHNVP were the minor metabolites with only a trace amount of 2‐OHNVP detected in some pharmacokinetic samples. No 8‐OHNVP was observed in all of the pharmacokinetic samples. In addition, the fragmentation for the four hydroxyl metabolite isomers is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of midazolam and its three hydroxy metabolites in human plasma by electron-capture gas chromatography without derivatization

Electron-capture gas chromatography was carried out to determine midazolam and its three hydroxy metabolites (1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam) in human plasma. The assay involves extraction from plasma, buffered to pH 9.3, into cyclohexane-dichloromethane (6:4) and analysis by gas chromatography. The use of an HP-17 cross-linked, capillary column makes derivatization unnecessary. The sensitivity of the method was 2-3 ng/ml for midazolam, 1-hydroxymethylmidazolam and 4-hydroxymidazolam, and 20 ng/ml for 1-hydroxymethyl-4-hydroxymidazolam. The extraction recovery of midazolam, 1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam was 99.3 +/- 2.4, 67.0 +/- 4.6, 92.7 +/- 4.7 and 28.7 +/- 6.3%, respectively. This gas chromatographic assay was used to assess the concentration-time profiles of midazolam and its metabolites in human plasma after rectal and intravenous administration of midazolam.     

Quantitation of pancuronium, 3-desacetylpancuronium, vecuronium, 3-desacetylvecuronium, pipecuronium and 3-desacetylpipecuronium in biological fluids by capillary gas chromatography using nitrogen-sensitive detection

A sensitive and specific capillary gas chromatographic (GC) assay was developed for the quantitation of the quaternary ammonium steroidal neuromuscular blocking drugs pancuronium (PANC), vecuronium (VEC) and pipecuronium (PIP), as well as the metabolites 3-desacetylpancuronium (3-desPANC) and 3-desacetylvecuronium (3-des VEC) in plasma, bile and urine; the putative metabolite 3-desacetylpipecuronium (3-des PIP) was extracted and quantitated only in urine. The procedure employed a single dichloromethane extraction of the iodide ion-pairs of the monoquaternary or bisquaternary ammonium compounds (including internal and external standards) from acidified, ether-washed biological fluid followed by the formation of stable O-tert.-butyldimethylsilyl derivatives at the 3-hydroxy steroidal position of the metabolites. An automated capillary GC system fitted with a nitrogen-sensitive detector and an integrator was then used to analyze and quantitate both parent compounds and their derivatized metabolites. Optimal extraction, derivatization and GC conditions, as well as short-term stability and recoveries of these drugs and metabolites in plasma, are reported. Electron ionization mass spectrometry combined with GC was used to confirm the identities of compounds eluted from the column. The assay demonstrated a 10(3)-fold linear range up to 5000 ng/ml for PANC, VEC, 3-des VEC and PIP, and lower limits of detection with adequate precision of 2 ng/ml for PANC, VEC and PIP, and 4 ng/ml for 3-des VEC; 3-des PANC was linear from 8 to 500 ng/ml while 3-des PIP was linear from 25 to 1000 ng/ml. The precision (coefficient of variation) of the calibration curves for underivatized drugs and their derivatized metabolites over the linear ranges was 2-20% and the reproducibility of the assay over a range of clinical concentrations of these drugs found in human plasma was 5-16% for PANC, 2-4% for VEC and 6-11% for PIP. No interferences were detected in the assay of plasma samples from 106 surgical patients.     

LC Method for the Determination of NPC1161, Primaquine and their Metabolites in Various Biological Systems

NPC1161 is an 8-aminoquinoline anti-malarial analog, which has a favorable toxicity profile relative to primaquine and other 8-aminoquinolines. High-performance liquid chromatographic method was developed for the determination of NPC1161, primaquine and their metabolites in biological samples in order to facilitate metabolic and pharmacokinetic studies. The method includes extraction of the unchanged drugs and their metabolites from the biological samples. Separation was achieved by reversed-phase chromatography on a C18 column with water–acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. Recoveries of NPC1161 and its metabolites were greater than 60% in various biological samples tested. No interference with the components of the biological material was observed. The detector response was linear with concentrations of NPC1161 and its metabolites (desalkyl NPC1161 and carboxy NPC1161) in the ranges from 0.5 to 80.0, 0.4–60.0 and 0.4–70.0 μg mL⁻¹, respectively. A mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of NPC1161 and its metabolites in biological samples. This method involved the use of the [M + H]⁺ions of NPC1161, C3 analog (internal std. for the assay), desalkyl NPC1161 and carboxy NPC1161 at m/z 434, 406, 349 and 449 in the positive ion mode with extractive ion monitoring (EIM). This method will have an important application in pharmacokinetic studies of NPC1161 and in understanding the mechanism of metabolism of this novel 8-aminoquinoline analog in more detail.     

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

Measurement of xenobiotics in saliva: is saliva an attractive alternative matrix? Case studies and analytical perspectives

The use of saliva for measuring xenobiotic concentrations has been practiced for a number of years. While the use of saliva has been generally reserved for the analysis of diagnostic and forensic/toxicology samples, attempts have been made to further enhance the value of saliva as an alternate matrix to those of plasma and serum. It is understood that saliva represents a handy tool for therapeutic drug monitoring (TDM) as it offers certain distinctive advantages. This scope of this review encompasses the following: (a) a comprehensive view of saliva as an alternate matrix for either plasma or serum to understand the pharmacokinetic/pharmacodynamic (PK/PD) characteristics; (b) an account of the factors contributing to the observed variability in salivary monitoring; (c) a tabular compilation of diverse case studies of xenobitoics belonging to different therapeutic classes with emphasis on assay methodology and applicable analytical/biopharmaceutical/pharmacokinetic findings; (d) relevant thoughts on assay procedures as they relate to salivary monitoring; and (e) some representative case studies highlighting the new thinking on the use of saliva outside of traditional TDM. Overall, based on the review, saliva represents a valuable TDM tool for a number of xenobiotics. While parent compound and phase I metabolite(s) for many xenobiotics have been generally quantifiable in saliva, phase II metabolites have not generally been detected in saliva. Therefore saliva samples could also be used to answer some specific PK/PD questions during the drug development process, if applicable. However, the development and validation of the assay in saliva needs to be carried out carefully with particular focus on proper sample collection, processing and storage to ensure the stability of the xenobiotics and with the same rigor as applied to plasma, serum and urine matrices.     

A fast method for the quantitation of key metabolites of the methionine pathway in liver tissue by high-resolution mass spectrometry and hydrophilic interaction ultra-performance liquid chromatography

We developed an assay for the extraction and simultaneous quantitation of five key metabolites of the methionine metabolic pathway in liver tissue. The metabolites included were 5′-methylthioadenosine, methionine, homocysteine, S-adenosyl-l-homocysteine, and S-adenosyl-l-methionine. The metabolites were extracted using a bead-based homogenization method, and quantitation was carried out using hydrophilic interaction chromatography and time-of-flight mass spectrometry. The extraction procedure was optimized by testing the effect of various solvent combinations. The chromatographic method was optimized for peak shape, signal intensity, and carry-over. With a total chromatographic run time of 5 min, this assay is suitable for the analysis of large sample sets. Time-of-flight mass spectrometry provided high mass accuracy which, combined with isotope pattern matching and use of chemical standards, guarantees high specificity. Moreover, by operating the mass spectrometer in enhanced duty cycle mode the signal strength for the analytes increased three- to tenfold in comparison with the generic full-scan mode. For quantitation, a matrix-spiked calibration method was used. The lowest analyte levels detected and quantified using our method were within the range of concentrations found in the liver. The inter-day coefficients of variance for the analytes were between 5 and 15 % in pooled tissue samples. Interestingly, the CVs between individual liver tissue aliquots were about twice as high. Additional experiments suggested that this higher variability was caused by uneven distribution of the analytes within the liver. In conclusion, an optimized and robust assay is now available for the extraction and quantification of key metabolites in the methionine metabolic pathway.     

High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.     

Quantitation of digitoxin and the bis- and monodigitoxosides of digitoxigenin in serum

A specific assay is described for measuring the concentration of digitoxin and the bis- and monoglycosides of digitoxigenin in serum. The procedure includes: (1) addition of a tracer amount of tritium labeled parent compound to the serum in order to measure percentage recovery; (2) solvent extraction to separate polar and non-polar metabolites; (3) reversed-phase thin-layer chromatography of the non-polar fraction to separate digoxigenins from digitoxigenins; (4) thin-layer chromatography to isolate digitoxin, and the bis- and monoglycosides of digitoxigenin; and (5) use of an 125I-radioimmunoassay to determine the concentration of the glycosides. Each of these three glycosides was administered intravenously to a normal subject, and the concentration of parent compound was measured in the serum at various times.     

A novel procedure for the determination of tricyclic antidepressants in plasma by use of high performance liquid chromatography.

A novel method for the determination of the antidepressant 3-(1-chloro-5-H-dibenzo [a,d] cycloheptene-5-ylidene)-N,N-dimethylpropylamine-N-oxide hydrochloride and its metabolites by use of high performance liquid chromatography was developed. The procedure is applicable to the assay of other similar drugs in biological samples. The method involves extraction of the unchanged drug and its metabolites from plasma, back-extraction into diluted phosphoric acid and re-extraction into an organic phase. Separation is performed on a silica gel column with an acidic mobile phase, containing sodium dodecyl sulfate as ion-pairing agent. The quantitation is carried out by UV detection. The procedure allows the determination of plasma levels down to about 5 ng/ml of the unchanged drug and its metabolites, respectively, when 1 ml of plasma is used. The plasma levels of two volunteers were determined after a single oral dose of the drug.