Modified gas chromatographic–mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma

A modified method for the determination of gacyclidine enantiomers in human plasma by GC–MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d₃-gacyclidine) as internal standard was developed. Following a single-step liquid–liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d₃-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (−)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (−)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) <14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r²>0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were <9% at 5, 10 and 20 ng/ml, and <16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d₃-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.     

Enantioselective analysis of propafenone in plasma using a polysaccharide-based chiral stationary phase under reversed-phase conditions

We present a method for the enantioselective analysis of propafenone in human plasma for application in clinical pharmacokinetic studies. Propafenone enantiomers were resolved on a 10-μm Chiralcel OD-R column (250×4.6 mm I.D.) after solid-phase extraction using disposable solid-phase extraction tubes (RP-18). The mobile phase used for the resolution of propafenone enantiomers and the internal standard propranolol was 0.25 M sodium perchlorate (pH 4.0)–acetonitrile (60:40, v/v), at a flow-rate of 0.7 ml/min. The method showed a mean recovery of 99.9% for (S)-propafenone and 100.5% for (R)-propafenone, and the coefficients of variation obtained in the precision and accuracy study were below 10%. The proposed method presented quantitation limits of 25 ng/ml and was linear up to a concentration of 5000 ng/ml of each enantiomer.     

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of

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-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate–methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (λ_ex 325 nm, λ_em 435 nm), a limit of detection of 0.1–0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4–0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86–110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence

A sensitive capillary electrophoretic method for the determination of carvedilol enantiomers in 100 μl of human plasma has been developed and validated. Carvedilol and the internal standard carazolol are isolated from plasma samples by liquid–liquid extraction using diethylether. A sensitive and selective detection is provided by helium–cadmium laser-induced fluorescence. The total analysis time is 17.5 min, about 30 min are needed for the sample preparation. The linearity of the assay ranges from 1.56 to 50 ng/ml per carvedilol enantiomer. The limits of quantification (LOQ) for the carvedilol enantiomers in 100 μl of human plasma are 1.56 ng/ml. The inter-day accuracy for R-carvedilol is between 95.8 and 103% (104% at LOQ) and for S-carvedilol between 97.1 and 103% (107% at LOQ); the inter-day precision values are between 3.81 and 8.64% (10.9% at LOQ) and between 5.47 and 7.86% (7.91% at LOQ) for R- and S-carvedilol, respectively. The small sample volume needed is especially advantageous for the application in clinical studies in pediatric patients. As an application of the assay concentration/time profiles of the carvedilol enantiomers in a 5-year-old patient receiving a test dose of 0.09 mg/kg carvedilol are reported.     

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid–liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R-didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated β-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.     

High-performance liquid chromatographic assay of mepivacaine enantiomers in human plasma in the nanogram per milliliter range

A method enabling quantification of R-(−)- and S-(+)-mepivacaine in human plasma in the low nanogram per milliliter range is described. The procedure involves extraction from plasma with diethyl ether, centrifugation, back-extraction into an acidified aqueous solution, washing with a mixture of pentane and isoamylalcohol, alkalinisation, followed by extraction with a mixture of n-pentane and isoamylalcohol. After evaporation of the organic phase, the residue is redissolved in the mobile phase used for the HPLC analysis, which consists of a 6.8:93.2 (v/v) isopropanol-sodium hydrogenphosphate buffer solution with the pH adjusted to 6.8 using phosphoric acid. The HPLC method has been described previously. Separation of the enantiomers is achieved with an α₁-AGP column and the UV detection wavelength is 210 nm. The minimal detectable concentration is ca. 3 ng/ml and the lower limit of quantification is 5 ng/ml for each enantiomer. For both enantiomers r is >0.9995 over the plasma enantiomeric concentration range of 10.5–1054 ng/ml.     

Direct enantioseparation of some β-adrenergic blocking agents using impregnated thin-layer chromatography

The resolution of (±)-atenolol, (±)-propranolol and (±)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure

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-lysine (0.5%) and

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-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile–methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (±)-atenolol and (±)-propranolol was 2.6 μg and for (±)-metoprolol, it was 0.26 μg.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

Liquid chromatographic determination of total celiprolol or (S)-celiprolol and (R)-celiprolol simultaneously in human plasma

A method has been developed for the determination of total celiprolol (sum of enantiomers) or the enantiomers (R)-celiprolol and (S)-celiprolol in plasma by high-performance liquid chromatography with UV and fluorescence detection. After extraction from alkalinized plasma with methyl-tert-butyl ether and back-extraction into 0.01 M HCl (for total celiprolol determination) or after evaporation of the organic phase and derivatisation with R(−)-1-(1-naphthyl)ethyl isocyanate (enantiomer determination), total celiprolol or its diastereomeric derivatives were chromatographed on a reversed-phase HPLC column with a mixture of acetonitrile and phosphate buffer pH 3.5 (+0.05% triethylamine). Acebutolol was used as internal standard. Linearity was obtained in the range of 5 to 2000 ng/ml for total and 2.5 to 500 ng/ml for enantiomer determination. Intra-day and inter-day variation was lower than 10%. The method can be applied for analysis of plasma samples obtained from patients treated with oral racemic celiprolol doses.     

Determination of the enantiomers of a novel 20,21-dinoreburnamenine derivative in rat plasma and brain by high-performance liquid chromatography using a chiral stationary phase

A high-performance liquid chromatographic method with solid-phase extraction was developed for the assay of the enantiomers of a novel 20,21-dinoreburnamenine derivative (RU 49041) in rat plasma and brain using a chiral stationary phase (Nucleosil Chiral 2) and ultraviolet detection. The limit of detection was 10 ng/ml (or ng/g) in both tissues and the intra-assay precision was satisfactory (plasma, ca. 5%; brain, ca. 1%). The pharmacokinetic profiles of the two enantiomers were determined following oral administration of the racemate (10 mg/kg). The results show that their pharmacokinetics are very different: whereas both enantiomers appear in the brain, only the 3α,16β-enantiomer is detected in plasma.     

Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography

An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α₁-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.     

Enantiomeric separation of metoprolol and α-hydroxymetoprolol by liquid chromatography and fluorescence detection using a chiral stationary phase

A sensitive and stereoselective high-performance liquid chromatographic assay for the determination of the enantiomers of metoprolol (R- and S-) and the diastereoisomers of α-hydroxymetoprolol (IIA, IIB) in plasma is reported. Chromatography involved direct separation of enantiomers using a Chirobiotic T bonded phase column (250×4.6 mm) and a mobile phase consisting of acetonitrile–methanol–methylene chloride–glacial acetic acid–triethylamine (56:30:14:2:2, v/v). Solid-phase extraction using silica bonded with ethyl group (C₂) was used to extract the compounds of interest from plasma and atenolol was used as the internal standard. The column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 225 and 310 nm, respectively. S-Metoprolol,R-metoprolol, IIB and IIA eluted at about 5.9, 6.7, 7.3 and 8.2 min without any interfering peaks. The calibration curve was linear over the range of 0.5 to 100 ng/ml for each isomer of metoprolol and 1 to 100 ng/ml for each isomer of α-hydroxymetoprolol (IIA & IIB). The mean intra-run accuracies were in the range of 96.2 to 114% for R-metoprolol, 94.0 to 111% for S-metoprolol, 90.2 to 110% for IIA, and 94.6 to 106% for IIB. The mean intra-run precisions were all in the range of 2.2 to 12.0% for R-metoprolol, 2.1 to 11.1% for S-metoprolol, 1.9 to 14.5% for IIA, and 3.2 to 11.0% for IIB. The lowest level of quantitation for the enantiomers of metoprolol was 0.5 ng/ml and 1.0 ng/ml for α-hydroxymetoprolol (IIA and IIB). The absolute recoveries for each analyte was ≥95%. The validated method accurately quantitated the enantiomers of parent drug and metabolite after a single dose of an extended release metoprolol formulation.     

High-performance liquid chromatographic determination of thiopentene enantiomers in sheep plasma

An HPLC method was developed to determine the plasma concentrations of R(+)- and S(−)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma samples were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm × 4 mm I.D.. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k′ values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(−)-pentobarbitone, R(+)-thiopentone, S(−)-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S(−)-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 μg/ml were, respectively, 4.8, 4.4 and 3.5% for R(+)-thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(−)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentene and 8.8, 7.4 and 9.6% for S(−)-thiopentone. Linearity over the standard range, 0.01–40 μg/ml, was shown by correlation coefficients> 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac-thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone eantiomers.     

High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids

Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(−)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane—isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (α = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always <9.8%. The lowest quantifiable concentration was 0.5 μg/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.     

Usefulness of 1,3 Beta-<Emphasis Type="SmallCaps">d</Emphasis>-Glucan Detection in non-HIV Immunocompromised Mechanical Ventilated Critically Ill Patients with ARDS and Suspected <Emphasis Type="Italic">Pneumocystis jirovecii</Emphasis> Pneumonia

Introduction

Pneumocystis jirovecii pneumonia (PCP) is a major cause of disease in immunocompromised individuals. Diagnosis is typically obtained by microscopy and/or PCR. For ambiguous PCR results, we evaluated the new biomarker 1,3-Beta-d-Glucan (BDG).

Methods

BDG serum levels were assessed and correlated to PCR results in immunosuppressed patients with ARDS.

Results

11 (22%) out of 50 patients had suspected PCP. APACHE II (26 vs. 24; p < 0.002), SOFA score (16 vs. 14; p < 0.010) and mortality rate (34 vs. 69% p < 0.004; 34 vs. 80% p < 0.003) were significantly altered in patients with positive (pPCR) and slightly positive (spPCR) PCJ PCR as compared to patients with no-PCP (nPCP). BDG levels were significantly lower in patients with nPCP (86; 30–315 pg/ml) than in patients with pPCR (589; 356–1000 pg/ml; p < 0.001) and spPCP (398; 297–516 pg/ml; p < 0.004) referring to the cutoff in this study for PCP of 275 pg/ml. An overall sensitivity (S) of 92% (95% CI 86–96%) and specificity (SP) of 84% (95% CI 79–85%) for PCP were found for the BDG Fungitell assay. In detail, S of 98% (95% CI 94–100%) and SP of 86% (95% CI 82–92%) for pPCP and S of 98% (95% CI 96–100%) and SP of 88% (95% CI 86–96%) for spPCO were found.

Conclusion

Serum BDG levels were strongly elevated in PCP, and the negative predictive value is high. BDG could be used as a preliminary test for patients with suspected PCP, especially in patients with slightly positive PCR results.

     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Stereoselective determination of fenfluramine enantiomers in rat liver microsomal incubates

An enantioselective assay for l- and d-fenfluramine in rat liver microsomal incubates was developed. The method involves extraction of fenfluramine from the microsomal incubates, and formation of fenfluramine diastereomeric derivatives with the chiral reagent S-(−)-N-trifluoroacetyl prolyl chloride. Separation and quantitation of the diastereomeric fenfluramine derivatives are carried out by a capillary gas chromatographic system with flame ionization detection. The assay is linear from 1 to 50 μg/ml for each enantiomer. The analytical method affords average recoveries of 92.28 and 96.44% for l- and d-fenfluramine, respectively. The limits of detection and quantitation for the method are 0.1 and 1.0 μg/ml for the l- and d-fenfluramine isomers, respectively. The reproducibility of the assay was <10% (RSD). The method allowed study of the depletion of l- and d-fenfluramine in rat liver microsomal incubates. The stereoselectivity of fenfluramine phase I metabolism was observed.