High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP⁺). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Analysis of amphetamine-type stimulants and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry

The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50-5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and -9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Automated solid-phase extraction and liquid chromatography-electrospray ionization-mass spectrometry for the determination of flunitrazepam and its metabolites in human urine and plasma samples

A sensitive and specific method using reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (LC-ESI-MS) has been developed for the quantitative determination of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DMF) and 3-hydroxyflunitrazepam (3-OHF) in biological fluids. After the addition of deuterium labelled standards of F,7-AF and N-DMF, the drugs were isolated from urine or plasma by automated solid-phase extraction, then chromatographed in an isocratic elution mode with a salt-free eluent. The quantification was performed using selected ion monitoring of protonated molecular ions (M+H(+)). Experiments were carried out to improve the extraction recovery (81-100%) and the sensitivity (limit of detection 0.025 ng/ml for F and 7-AF, 0.040 ng/ml for N-DMF and 0.200 ng/ml for 3-OHF). The method was applied to the determination of F and metabolites in drug addicts including withdrawal urine samples and in one date-rape plasma and urine sample.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone ®) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C₁₈, 250x4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m² of mitoxantrone by intravenous infusion for 10 min.     

Identification of a new functional target of haloperidol metabolite: implications for a receptor-independent role of 3-(4-fluorobenzoyl) propionic acid

Haloperidol, a dopamine D2 receptor blocker, is a classical neuroleptic drug that elicits extrapyramidal symptoms. Its metabolites include 3-(4-fluorobenzoyl) propionic acid (FBPA) and 4-(4-chlorophenyl)-4-piperidinol (CPHP). Until now, the biological significance of these metabolites has remained largely unknown. Here, we report that the administration of FBPA to mice effected a suppression of locomotor activity and induced catalepsy in a manner similar to that observed with haloperidol, whereas CPHP had no significant effects. Neither of these two metabolites, however, exhibited any ability to bind to the dopamine D2 receptor. FBPA blocked dopamine-induced extracellular signal-regulated kinase 1/2 phosphorylation, and it specifically affected mitogen-activated protein kinase kinase (MEK)1/2 activity in hippocampal HN33 cells. Moreover, FBPA was capable of direct interaction with MEK1/2, and inhibited its activity in vitro. We demonstrated the generation of haloperidol metabolites within haloperidol-treated cells by mass spectrometric analyses. Collectively, our results confirm the biological activity of FBPA, and provide initial clues as to the receptor-independent role of haloperidol.     

Determination of diphenylpyraline in plasma and urine by high-performance liquid chromatography

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Simultaneous determination of haloperidol and bromperidol and their reduced metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography

This study describes a new simultaneous determination of haloperidol and bromperidol and their reduced metabolites by modification of automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1ml of plasma using chloroform-hexane (30:70 (v/v)), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C(18) analytical column for separation. The mobile phases consisted of phosphate buffer (0.02M, pH 4.6), perchloric acid (60%) and acetonitrile (54:1:45 (v/v)) and was delivered at a flow-rate of 0.6ml/min. The peak was detected using a UV detector set at 215nm. The method was validated for the concentration range 1-100ng/ml, and good linearity (r >0.999) was confirmed. Intra-day coefficient variations (CVs) for haloperidol, reduced haloperidol, bromperidol and reduced bromperidol were less than 2.5, 3.1, 2.4 and 2.5%, respectively. Inter-day CVs for corresponding compounds were 3.9, 5.1, 2.6 and 4.4%, respectively. Relative errors ranged from -5 to 10% and mean recoveries were 96-100%. The limit of quantification was 1.0ng/m for each compound. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring, particularly in patients receiving both haloperidol and bromperidol.     

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Determination of methylnaltrexone in clinical samples by solid-phase extraction and high-performance liquid chromatography for a pharmacokinetics study

A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 microm, 150x3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n = 10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C(max) = 110 (+/-55) and 287 (+/-101) ng/ml and t(max) = 16.7 (+/-10.8) and 20.0 (+/-9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.     

Kinetics of kavain and its metabolites after oral application

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC-DAD) assay method for the simultaneous determination of kavain and its main metabolites (p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC-MS or LC-MS-MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix

A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r2=0.999) was acceptable over the range of 10-5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or -20 degrees C.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.