Gas chromatographic–mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30°C, DCBs were absorbed for 15 min by a 100-μm polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography–mass spectrometry (GC–MS). By setting the initial column oven temperature at 20°C, the three isomers were resolved at the baseline level. p-Xylene-d₁₀ was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2–10.7% were found at blood concentrations of 1.0 and 10 μg/ml. Each compound was detectable at a level of at least 0.02 μg per 1 g of whole blood (by full mass scanning). HS-SPME–GC–MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.     

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring (GC–MS–EI-SIM). Deuterated lidocaine (d₁₀-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d₁₀-lidocaine (I.S.) was heated at 120°C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS system. The calibration curves showed linearity in the range of 0.1–20 μg/g for lidocaine and mepivacaine, 0.5–20 μg/g for bupivacaine and 1–20 μg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico–legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 μg/g, respectively.     

High-temperature solid-phase microextraction procedure for the detection of drugs by gas chromatography–mass spectrometry

High-temperature headspace solid-phase microextraction (SPME) with simultaneous (“in situ”) derivatisation (acetylation or silylation) is a new sample preparation technique for the screening of illicit drugs in urine and for the confirmation analysis in serum by GC–MS. After extraction of urine with a small portion of an organic solvent mixture (e.g., 2 ml of hexane–ethyl acetate) at pH 9, the organic layer is separated and evaporated to dryness in a small headspace vial. A SPME-fiber (e.g., polyacrylate) doped with acetic anhydride–pyridine (for acetylation) is exposed to the vapour phase for 10 min at 200°C in a blockheater. The SPME fiber is then injected into the GC–MS for thermal desorption and analysis. After addition of perchloric acid and extraction with n-hexane to remove lipids, the serum can be analysed after adjusting to pH 9 as described for urine. Very clean extracts are obtained. The various drugs investigated could be detected and identified in urine by the total ion current technique at the following concentrations: amphetamines (200 μg/l), barbiturates (500 μg/l), benzodiazepines (100 μg/l), benzoylecgonine (150 μg/l), methadone (100 μg/l) and opiates (200 μg/l). In serum all drugs could be detected by the selected ion monitoring technique within their therapeutic range. As compared to liquid–liquid extraction only small amounts of organic solvent are needed and larger amounts of the pertinent analytes could be transferred to the GC column. In contrast to solid-phase extraction (SPE), the SPME-fiber is reusable several times (as there is no contamination by endogenous compounds). The method is time-saving and can be mechanised by the use of a dedicated autosampler.     

Headspace solid-phase microextraction and gas chromatographic–mass spectrometric screening for volatile hydrocarbons in blood

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic–mass spectrometric (GC–MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC–MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 μg of toluene-d₈ at −5°C were adsorbed to a 100-μm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC–MS method. The lower detection limits of all the compounds were 0.01 μg per 1 g of blood. Linearities (r²) within the range 0.01 to 4 μg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 μg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.     

Application of solid-phase microextraction and gas chromatography–mass spectrometry for the determination of chlorophenols in urine

This study investigated the feasibility of applying solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry to analyze chlorophenols in urine. The SPME experimental procedures to extract chlorophenols in urine were optimized with a polar polyacrylate coated fiber at pH 1, extraction time for 50 min and desorption in GC injector at 290°C for 2 min. The linearity was obtained with a precision below 10% R.S.D. for the studied chlorophenols in a wide range from 0.1 to 100 μg/l. In addition, sample extraction by SPME was used to estimate the detection limits of chlorophenols in urine, with selected ion monitoring of GC–MS operated in the electron impact mode and negative chemical ionization mode. Detection limits were obtained at the low ng/l levels. The application of the methods to the determination of chlorophenols in real samples was tested by analyzing urine samples of sawmill workers. The chlorophenols were found in workers, the urinary concentration ranging from 0.02 μg/l (PCP) to 1.56 μg/l (2,4-DCP) depending on chlorophenols. The results show that trace chlorophenols have been detected with SPME–GC–MS in the workers of sawmill where chlorophenol-containing anti-stain agents had been previously used.     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg, -Trp, mePhe]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, -Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Assay method for the perfluorooctyl bromide (perflubron) in rat blood by gas chromatography–mass spectrometry

This paper describes a GC–MS method (SIM mode) for the analysis of perfluorooctyl bromide (perflubron, I) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Following destruction of the emulsion by addition of ethanol, the analytical procedure involves a liquid–liquid extraction with 1,1,2-trichlorotrifluoroethane. The bis(F-butyl)ethene (II) was used as internal standard. Observed retention times were 3.22 min for I and 2.32 min for II. Two calibration curves were used; linear detection responses were obtained for concentrations ranging from 0.009 to 0.9 mg/ml and from 0.9 to 13.5 mg/ml. The extraction efficiency averaged 50% for I and 93% for II. Precision ranged from 0.7 to 14%, and accuracy was between 91 and 109%. The limit of quantification was 9 μg/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay method for use in pharmacokinetic studies.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea in plasma

A facile, sensitive and highly specific HPLC method for assaying 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extraction with tert.-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I.S.). Following evaporation of the organic solvent, the extract was reconstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded onto a 4 μm Nova-Pak C₁₈ column (15 cm×3.9 mm), which was preceded by a 7 μm Brownlee RP-18 precolumn (1.5 cm×3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol-0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of the effluent was monitored at 240 nm. A flow-rate of 1.0 ml/min was used for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. An automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mobile phase to the analytical column, thereby preventing the apparent build-up of extractable, strongly retained, UV-absorbing components present in mouse and dog plasma. Operating in this manner, more than 100 samples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determination of SarCNU in human plasma by simply decreasing the eluent flow-rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximately 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-μl sample volume, the lowest concentration of SarCNU included in the plasma standard curves, 0.10 μg/ml, was quantified with a 7.8% R.S.D. (n=27) over a 2 month period. Plasma standards, with concentrations of 0.26 to 5.1 μg/ml, exhibited R.S.D. values ranging from 1.3 to 4.7%. Thermospray-ionization MS detection was used to definitively establish the specificity of the method. The sensitivity of the assay was shown by application to be more than adequate for characterizing the plasma pharmacokinetics of SarCNU in mice.     

High-performance liquid chromatographic–tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells

We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic–tandem mass spectrometric method (HPLC–MS–MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC). The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min. Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA). BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK). The detection limits for ethionamide were 0.05 μg/ml for plasma, and 0.005 μg/ml for BAL supernatants and alveolar cell suspensions.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

High-performance size-exclusion chromatographic analysis of dextran–methylprednisolone hemisuccinate in rat plasma

A size exclusion chromatographic method is presented for the measurement of the concentrations of a macromolecular prodrug of methylprednisolone (MP), dextran–methylprednisolone succinate (DEX–MPS), in rat plasma. After precipitation of the plasma (100 μl) proteins with perchloric acid, the samples are injected into a size exclusion column with a mobile phase of water:acetonitrile:glacial acetic acid (75:25:0.2) and a flow-rate of 1 ml/min. The DEX–MPS conjugate, detected at 250 nm, elutes at a retention time of 6.5 min, free of endogenous peaks. Excellent linear relationships (r²=0.997) were found between the detector response and the concentrations of DEX–MPS in the range of 2–100 μg/ml (MP equivalent), with intra- and inter-run C.V.s of <6% and error values of <5%. The application of the assay was also demonstrated by measurement of the plasma concentrations of DEX–MPS after single 5 or 10 mg/kg doses of the conjugate administered intravenously to rats.     

Gas chromatographic–mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples

A simple and rapid method is described for the GC–MS determination of 4-nonylphenols (NOs) and 4-tert-octylphenol (OC) in biological samples. The NOs and OC in the sample are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR column clean-up, the sample extract is analyzed by GC–MS in the selected ion monitoring (SIM) mode. Average recoveries in pale chub (fish) and corbicula (shellfish) are 86.0 and 93.4% for NOs, and 95.8 and 96.4% for OC, respectively, spiked at the levels of 1.0 μg of NOs and 0.1 μg of OC per 5 g of fish and shellfish samples. The detection limits are 20 ng/g for NOs and 2 ng/g for OC.     

Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring

Plasma phenobarbital (PB) concentrations in rat offspring were determined using a 9 μl capillary by high-performance liquid chromatography (HPLC). Capillary plasma which was put into a Bond Elut^® cartridge column by using 1 ml of 0.01 M KH₂PO₄ was applied to the column with 50 μl of 2 μg/ml of acetanilide (internal standard, I.S.). After washing the column, PB and I.S. were eluted with methanol and injected into the HPLC system. There were excellent linear correlation between the amount of PB and length of the capillary at three different concentrations. Calibration for PB was linear in the range of 0–50 μg/ml. The coefficients of variation were 3.4–5.0% and 5.9–7.5% in the within-day and between-day assays, respectively. The extraction recovery rates were 87.5–105.4%. By this method, it was possible to measure plasma PB concentrations in rat offspring without killing. These results suggested that this method is very useful to determine the plasma PB concentration derived from mother’s milk in newborn rats.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.