Serotonergic neurotransmission mediates hypothermia induced by the N-phenylpiperazine antipsychotic prototypes LASSBio-579 and LASSBio-581

Previous studies have demonstrated that LASSBio-579 and LASSBio-581, two N-phenylpiperazine derivatives designed for the treatment of schizophrenia, are presynaptic dopamine D(2) receptor agonists that induce a hypothermic effect in mice that is not mediated by dopamine receptor activation. The aim of the present study was to investigate possible serotonergic mechanisms underlying hypothermia induced by LASSBio-579 and LASSBio-581 in CF1 mice. The reduction in core temperature was dose-dependent (15-60 mg/kg, i.p.) and occurred by the oral route (30 mg/kg). Pretreatment with haloperidol (4 mg/kg, i.p.) resulted in a synergistic hypothermic effect. Pretreatment with (+/-)DOI (0.25 mg/kg, i.p.), a serotonin 5-HT(2A/C) receptor agonist, reduced the hypothermic effect induced by LASSBio-579 and LASSBio-581 at 15 and 30 mg/kg, i.p. In contrast, (+/-)DOI enhanced the hypothermia induced by both compounds at 60 mg/kg, i.p. The serotonin 5-HT1A antagonist WAY 100635 (0.05 mg/kg, s.c.) abolished the hypothermia induced by LASSBio-579 and diminished the hypothermia induced by LASSBio-581. Pretreatment with LASSBio579 (30 and 60 mg/kg, i.p.) and LASSBio-581 (60 mg/kg, i.p.) reduced the number of head-twitches induced by (+/-)DOI (2.5 mg/kg, i.p.). The ear-scratch response induced by (+/-)DOI was inhibited by both LASSBio-579 and LASSBio-581 at 60 mg/kg, i.p. These results indicate that LASSBio-579 and LASSBio-581 have mechanisms of action through the serotonergic neurotransmitter system.     

CNS-selective noncompetitive cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline

LASSBio-767 [(−)-3-O-acetyl-spectaline] and LASSBio-822 [(−)-3-O-tert-Boc-spectaline] were recently described as cholinesterase inhibitors derived from the natural piperidine alkaloid (−)-spectaline, obtained from the flowers of Senna spectabilis (Fabaceae). We investigated their mechanism of inhibition of acetylcholinesterase and their efficacy in reversing scopolamine-induced amnesia. Competition assays with the substrate acetylthiocholine showed a concentration-dependent reduction in rat brain cholinesterase V_max without changes in apparent K_m. The kinetic data for LASSBio-767 and LASSBio-822 were best fit by a model of simple linear noncompetitive inhibition with K_i of 6.1 μM and 7.5 μM, respectively. A dilution assay showed a fast and complete reversal of inhibition, independent of incubation time. Simulated docking of the compounds into the catalytic gorge of Torpedo acetylcholinesterase showed interactions with the peripheral anionic site, but not with the catalytic triad. Anti-amnestic effects in mice were assessed in a step-down passive avoidance test and in the Morris water maze 30 min after injection of scopolamine (1 mg/kg i.p.). Saline, LASSBio-767, or LASSBio-822 was administered 15 min before scopolamine. Both compounds reversed the scopolamine-induced reduction in step-down latency at 0.1 mg/kg i.p. LASSBio-767 reversed scopolamine-induced changes in water maze escape latency at 1 mg/kg i.p. or p.o., while its cholinergic side effects were absent or mild up to 30 mg/kg i.p. (LD₅₀ above 100 mg/kg i.p.). Thus, the (−)-spectaline derivatives are potent cholinergic agents in vivo, with a unique profile combining noncompetitive cholinesterase inhibition and CNS selectivity, with few peripheral side effects.     

Synthesis and Evaluation of L-Glutamic acid Analogs as Potential Anticancer Agents

Four N-(benzenesulfonyl)-L-glutamic acid bis(p-substituted phenylhydrazides) were synthesized and evaluated for anticancer activity in vitro in DU-145 and PC-3 prostate cancer and in COLO-205 colon cancer cell lines by MTT assay. The analog with the nitro group substitution exhibited potent activity (% Inhibition 84.7 and 72.0 in DU-145 and PC-3 respectively at 80 μg/ml concentration). Another series of substituted 1-(benzenesulfonyl)-5-oxopyrrolidine 2-carboxamides (11a-f) were synthesized and evaluated for anticancer activity in vitro in colon (COLO-205), breast (Zr-75-1) and prostate (PC-3) cancer cell lines by MTT assay using adriamycin as standard. Test compounds 11a-c showed potent activity (% Inhibition 61.2 to 79.2 at 20 μg/ml and 67.2 to 87.2 at 40 μg/ml) in PC-3 cell line which is superior to the activity of Adriamycin. In comparison compounds 11d-f were less potent. In Zr-75-1 cell line 11a-e showed % inhibition ranging from 32.4 to 54.9 at 10 μg/ml concentration while in COLO-205 cell line 11a-f showed poor activity.     

High-performance liquid chromatographic analysis of a selective cyclooxygenase-1 inhibitor SC-560 in rat serum: application to pharmacokinetic studies

A method of analysis of SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism. A simple high-performance liquid chromatographic method was developed for simultaneous determination of SC-560 and other products of metabolism in rat serum. Serum (0.1 ml) was precipitated with acetonitrile after addition of the internal standard, testosterone 17-propionate. Separation was achieved on a C₈ column with UV-detection at 240 nm. The calibration curve was linear ranging from 0.02 to 100 μg/ml. The mean recovery was >86.7%. Precision of the assay was <10% (R.S.D.%), and was within 15% at the limit of quantitation (20 ng/ml). Bias of the assay was lower than 15.5%. The limit of detection was 10 ng/ml for a 0.1 ml sample. The assay was applied successfully to the in vivo kinetic study of SC-560 in rats.     

LC determination of dinitrosopiperazine in simulated gastric juice

A simple and specific reversed phase HPLC method for the determination of dinitrosopiperazine in simulated gastric juice using UV detection was reported. The chromatographic resolution of the analyte and the internal standard isosorbide dinitrate was performed without extraction from the gastric juice on a reversed phase ODS column. Isocratic elution was carried out with methanol–0.02 M sodium dihydrogen phosphate (60:40 v/v, pH 3.0) at a flow rate of 1.0 ml min⁻¹ with UV detection at 238 nm. The calibration graph was linear over the concentration range 0.072–2.88 μg ml⁻¹ of dinitrosopiperazine with minimum detectability (S/N=2) of 0.01 μg ml⁻¹ (8×10⁻⁸ M). Inter-day and intra-day precisions calculated as% RSD were in the range 0.32–0.38% and 0.19–0.25% respectively. Inter-day and intra-day accuracies calculated as% error were in the range 0.18–0.21 and 0.08–0.11% respectively. The proposed method was successfully applied to the study of the possible in–vivo production of DNPZ under the standard nitrosation conditions recommended by WHO.     

Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's Wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol–water (80:20, v/v) containing 5% HP-β-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 °C, were injected into a C₁₈ column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5–2.5 μg/ml (hypericin), 0.35–1.6 μg/ml (pseudohypericin) and 5–50 μg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were 4.4%, 5.4%, and 2.8%, respectively; inter-day CVs were 5.8%, 4.9%, and 2.5%, respectively. This method may be applied for the routine standardization of St. John's Wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.     

HPLC quantification of two isomeric triterpenic acids isolated from Mitracarpus scaber and antimicrobial activity on Dermatophilus congolensis

Oleanolic (OA) and ursolic acids (UA) were isolated for the first time from the alcoholic extract of Mitracarpus scaber possessing antimicrobial effects on Dermatophilus congolensis. These two triterpenic acids were also active (MIC 15 μg/ml) on this causative agent of dermatophilosis in African animals.

To quantify OA and UA in M. scaber, a new, simple and rapid high-performance liquid chromatography (HPLC) method compatible with MS detection was developed and validated. The mobile phase acetonitrile:H₂O (85:15, v/v) was pumped through a C18 octadecylsilyl silica column at a flow rate of 0.6 ml/min and the eluate was monitored at 215 nm. The calibration curves constructed between 0.5 and 10 μg/ml showed linear relationships with good R² values. The developed method was precise and reproducible with relative standard deviations (RSD) for these two active constituents between 0.22–2.06% (intraday) and 1.61–3.72% (interday) for concentrations from 0.5 to 6 μg/ml. Limits of detection and quantification were, respectively, 0.2 and 0.5 μg/ml.     

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

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

UV and Three Derivative Spectrophotometric Methods for Determination of Ezetimibe in Tablet Formulation

UV, first, second and third derivative spectrophotometric methods have been developed for the determination of ezetimibe in pharmaceutical formulation. The solutions of standard and sample were prepared in methanol. For the first method, UV spectrophotometry, the quantitative determination of the drug was carried at 233 nm and the linearity range was found to be 6-16 μg/ml. For the first, second and third derivative spectrophotometric methods the drug was determined at 259.5 nm, 269 nm and 248 nm with the linearity ranges 4-14 μg/ml, 4-14 μg/ml and 4-16 μg/ml. The calibration graphs constructed at their wavelength of determination were found to be linear for UV and derivative spectrophotometric methods. All the proposed methods have been extensively validated. The described methods can be readily utilized for the analysis of pharmaceutical formulation. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations.     

Effect of the mycotoxin, penicillic acid, on Tetrahymena pyriformis

The biological effects of penicillic acid, a mycotoxin produced by several species of Penicillium and Aspergillus in an eukaryotic system, was studied using Tetrahymena pyriformis HSM as a model. Growth was inhibited 54% by 15 μg of penicillic acid per ml of growth medium. Total inhibition of T. pyriformis growth occurred in the presence of 50 μg of penicillic acid per ml. Within 24 hr of the addition of penicillic acid to the medium, there was a measurable dose-dependent decrease in the population density of cells and an increase in generation time. A dose-related inhibition of T. pyriformis growth over the range of 5–25 μg penicillic acid per ml of growth medium was seen when cell counts were obtained at 33 hr; such data indicated a potential bioassay for penicillic acid employing this protozoan. Cell respiration was only marginally affected by this mycotoxin. Total cellular DNA decreased in the presence of 5 μg of penicillic acid per ml whereas higher concentrations (10 and 25 μg/ml of growth medium, respectively) were required before a decrease in RNA or protein was observed. Cells exposed to 20 μg of penicillic acid per ml had normal morphology and size distribution and the cilia beat normally. An unidentified compound, possibly a metabolite of penicillic acid, was detected in the growth medium containing viable T. pyriformis.     

Simultaneous determination and validation of antimicrobials in plasma and tissue of actinomycetoma by high-performance liquid chromatography with diode array and fluorescence detection

A simple, precise, and reliable chromatographic method was developed for the simultaneous determination in plasma and infected tissue of five antimicrobials proposed for the treatment of actinomycotic mycetoma: amoxicillin, trimethoprim, linezolid, sulfamethoxazole and garenoxacin. Separation of the analytes was achieved on an Atlantis dC18 column (150 mm × 4.6 mm, ID 5 μm) with a mobile phase composed of acetonitrile and trifluoroacetic acid (ATF) 0.1% (v/v) using a gradient program. The detection was carried out using a diode array detector at 254 nm and in a fluorescence detector at wavelengths of excitation and emission of 292 nm and 392 nm for linezolid and sulfamethoxazole, and 292 nm and 408 nm for garenoxacin, respectively. The intraday precision was in the range of 0.7–15% of relative standard deviations (%R.S.D.) for plasma and 1–18% for tissue. Linearity range was from 2.4 to 20 μg/ml for amoxicillin, 0.3 to 20 μg/ml for trimethoprim, sulfamethoxazole and linezolid, and 0.3 to 10 μg/ml for garenoxacin. Acetonitrile was used to precipitate proteins from plasma. Recoveries in plasma ranged from 71% to 118% and in infected tissue from 78% to 122%. Limits of detection (LODs) were 1.2 and 0.5 μg/ml for amoxicillin in plasma and tissue, respectively and 0.15 and 1.2 μg/ml in plasma and tissue, respectively for the other antimicrobials. The method can be applied for individual or simultaneous determination of the antimicrobials in plasma and tissue of mouse infected with actinomycetoma.     

Estimation of Amlodipine Besylate, Valsartan and Hydrochlorothiazide in Bulk Mixture and Tablet by UV Spectrophotometry

A simple, precise, accurate and economic simultaneous UV spectrophotometric method has been developed for the estimation of amlodipine besylate, valsartan and hydrochlorothiazide in combination in bulk mixture and tablet. The estimation was based upon measurement of absorbance at absorbance maxima of 359 nm, 317 nm and 250 nm for amlodipine besylate, hydrochlorothiazide and valsartan in methanol, respectively in bulk mixture and tablet. The Beer Lambert''s law obeyed in the concentration range 5-25 μg/ml, 10-50 μg/ml and 5-25 μg/ml for amlodipine besylate, hydrochlorothiazide and valsartan, respectively. The estimation of bulk mixture and tablet was carried out by simultaneous equation, Q-analysis and area under curve method for estimation of amlodipine besylate and hydrochlorothiazide and standard curve method for estimation of valsartan. The results were found to be in the range of 99.6±1.52% to 102±0.51%. Method was validated with respect to specificity, linearity, range, accuracy, precision, LOD, LOQ, robustness, ruggedness and can be applied for routine analysis of tablet dosage forms.     

Genotoxic, mutagenic and cytotoxic effects of the commercial dye CI Disperse Blue 291 in the human hepatic cell line HepG2

Textile dyes are discarded into the aquatic ecosystem via industrial effluents and potentially expose humans and local biota to adverse effects. The commercial dye CI Disperse Blue 291 which contains the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2), was tested for genotoxicity and cytotoxicity in the human hepatoma cell line HepG2, using the comet assay, micronucleus (MN) test and a cell viability test. Five different concentrations of the test compound were examined: 200 μg/ml, 400 μg/ml, 600 μg/ml, 800 μg/ml and 1000 μg/ml. An increase in comet tail length and in the frequency of MN was detected with exposure of cells to concentrations of the commercial dye from 400 μg/ml. Furthermore, the dye was found to decrease cell viability. The results of this study demonstrate for the first time the genotoxic and mutagenic effects of the dye CI Disperse Blue 291 in mammalian cells, thus stressing the need to develop non-mutagenic dyes and to invest in improving the treatment of effluents. These measures will help to prevent harmful effects that these compounds can have on humans and aquatic organisms that come in contact with them.     

Stereospecific high-performance liquid chromatographic validation of homoeriodictyol in serum and Yerba Santa (Eriodictyon glutinosum)

A stereospecific method of analysis of racemic homoeriodictyol (eriodictyol 3′-methyl ether) in biological fluids is necessary to study pharmacokinetics and disposition in fruits and herbs. A simple high-performance liquid chromatographic method was developed for the determination of homoeriodictyol enantiomers. Separation was achieved in a Chiralcel^® OJ-RH column with UV-detection at 288 nm. The standard curves in serum were linear ranging from 0.5 to 100.0 μg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was <15%, and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of homoeriodictyol enantiomers in rats and to the quantification of homoeriodictyol enantiomers in Yerba Santa (Eriodictyon glutinosum).     

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 μg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were −12.58 and −0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 μg/ml. 3-OHQ formation complied with Michaelis–Menten kinetics and the mean values±S.D. of K_m and V_max were 36.98±4.62 μg/ml and 321.39±3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.     

RP-HPLC Estimation of Ramipril and Telmisartan in Tablets

A rapid high performance liquid chromatographic method has been developed and validated for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A Genesis C18 column having dimensions of 4.6×250 mm and particle size of 5 μm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid): methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluents were monitored at 210 nm. The selected chromatographic conditions were found to effectively separate ramipril (R_t: 3.68 min) and telmisartan (R_t: 4.98 min) having a resolution of 3.84. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation. Linearity for ramipril and telmisartan were found in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml, respectively. The percentage recoveries for ramipril and telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of detection and the limit of quantitation for ramipril was found to be 0.5 μg/ml and 1.5 μg/ml respectively and for telmisartan was found to be 1.5 μg/ml and 3.0 μg/ml, respectively. The method was found to be robust and can be successfully used to determine the drug content of marketed formulations.     

Separation and determination of four active components in medicinal preparations by flow injection-capillary electrophoresis

A simple, rapid and accurate method for the separation and determination of paracetamol (Par), pseudoephedrine hydrochloride (Pse), dextromethorphan hydrobromide (Dex) and chlorphenamine hydrogen maleate (Chl) was developed by combination of flow injection and capillary zone electrophoresis for the first time. The analysis was carried out using an unmodified fused-silica capillary (75 mm × 75 μm i.d. × 375 μm o.d., effective separation length of 45 mm) and direct ultraviolet detection at 214 nm, 1.0 kV applied voltage. The optimized running buffer composed of 75 mM sodium borate–15% (v/v) acetonitrile (ACN) (pH^* 9.30) was applied for the separation of the four analytes. The separation was achieved in 4.5 min. The sample throughput rate could reach up to 19 h⁻¹. The repeatability (defined as relative standard deviation) was 0.6%, 1.0%, 2.1%, 1.9% with peak height evaluation and 0.7%, 1.8%, 0.7%, 1.1% with peak area evaluation for Par, Pse, Dex and Chl, respectively. The limits of detection (S/N = 3) were 0.22 μg/ml, 0.29 μg/ml, 0.42 μg/ml and 0.70 μg/ml for Par, Pse, Dex and Chl, respectively. The method was successfully applied to determine the four compounds in three cold medicines with recoveries in the range of 97.18–105.15%.     

Simultaneous determination of pyrimethamine, sulfadiazine and N-acetyl-sulfadiazine in plasma for monitoring infants in treatment of congenital toxoplasmosis

A method for the simultaneous determination of pyrimethamine, sulfadiazine and its metabolite N-acetyl-sulfadiazine in small plasma samples from neonates in treatment for congenital toxoplasmosis has been developed. In this method only 25 μl of plasma is used and a simple sample preparation based on protein precipitation and centrifugation provides highly reliable data as the recovery is about 100% and the precision is good. The analysis is performed using high performance liquid chromatography with UV and mass spectrometric (MS) detection. Pyrimethamine was found to give a linear response using MS detection in the range 0.02–5 μg/ml. Sulfadiazine and its metabolite N-acetyl-sulfadiazine were preferably analysed by UV at 269 nm in the concentration ranges 0.2–200 μg/ml for sulfadiazine and 0.2–50 μg/ml for N-acetyl-sulfadiazine.     

Development and validation of a LC-MS/MS method with electrospray ionization for determination of LASSBio-579 in rat plasma

A simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of LASSBio-579 in plasma rat, using fluconazole as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water (80:20, v/v), containing 0.4mM ammonium hydroxide and 0.2 mM acetic acid at a flow rate of 1.0 ml/min (split ratio 1:5). A Micromass triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the positive mode. Plasma samples were deproteinized with acetonitrile (1:2) and 50 microl of the supernatant were injected into the system. The retention times of LASSBio-579 and IS were approximately 4.7 and 2.4 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 30-2000 ng/ml with determination coefficient >0.98. The lower limit of quantification was 30 ng/ml. The accuracy of method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 13.5% and 6.4%, respectively. The applicability of the LC-MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after intraperitoneal administration of LASSBio-579 to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of LASSBio-579 in pre-clinical pharmacokinetic studies.     

A capillary GC-MS method for analysis of phenytoin and [C3]-phenytoin from plasma obtained from pulse dose pharmacokinetic studies

Stable isotope analogues of phenytoin are useful for pulse dose pharmacokinetic studies in epilepsy patients. A simultaneous assay was developed to quantitate phenytoin (5,5-diphenylhydantoin) and its stable isotope analogue [¹³C₃]-phenytoin (5,5-diphenyl-2,4,5-¹³C₃-hydantoin) from plasma. Quantitation was achieved by GC-MS analysis of liquid/liquid extracted plasma samples, with [²H₁₀]-phenytoin (5,5-di(pentadeuterophenyl)-hydantoin) as an internal standard. The total coefficients of variance (C.V._t) were <7% for phenytoin (2.5–40 μg ml⁻¹) and <10.3% for [¹³C₃]-phenytoin (0.1–6.0 μg ml⁻¹). The accuracy of the assay varied from 87.8–100.1% (phenytoin, 2.5–40 μg ml⁻¹) and 89.6–116.3% ([¹³C₃]-phenytoin, 0.02–6.0 μg ml⁻¹). The assay was tested under in vivo conditions by administration of a pulse dose of the stable isotope analogue to a single rat dosed to steady-state with fosphenytoin, a phenytoin prodrug. The results of the in vivo experiment demonstrate the usefulness of this assay for future pharmacokinetic studies in special population epilepsy patients.