Stereoselective inhibition of pindolol renal clearance by cimetidine in humans.

There are few data on whether differences exist in the renal tubular secretion of enantiomers and no data on whether inhibition of renal secretion of individual enantiomers is stereoselective. Pindolol was used as a probe drug because it is used clinically as a racemic mixture of R-(+) and S-(-) enantiomeric forms and is highly secreted by the proximal tubules of the kidney. Eight young healthy subjects received a single 15 mg oral dose of racemic pindolol with and without 400 mg cimetidine twice daily. The area under the plasma concentration-time curve of both R-(+)- and S-(-)-pindolol were significantly (p less than 0.01) increased by cimetidine from 234 +/- 90 (mean +/- SD) to 344 +/- 78 ng/ml.hr for R-(+)-pindolol and from 209 +/- 73 to 288 +/- 69 ng/ml.hr for S-(-)-pindolol. The renal clearance of R-(+)-pindolol (170 +/- 55 ml/min) was significantly (p less than 0.05) less than that for S-(-)-pindolol (222 +/- 66 ml/min). Cimetidine significantly (p less than 0.01) reduced the renal clearances of R-(+)-pindolol to 104 +/- 18 ml/min and for S-(-)-pindolol to 155 +/- 38 ml/min. The enantiomer with the higher renal clearance [S-(-)-pindolol] had its renal clearance reduced less by cimetidine (26% versus 34%, p less than 0.05). Cimetidine appears to have a stereoselective action on the active transport system for organic cations in the proximal tubule.

Hepatic disposition of electrophilic acyl glucuronide conjugates.

Acyl glucuronides are a unique class of electrophilic metabolites, capable of non-enzymatic reactions including acylation and/or glycation of endogenous macromolecules, hydrolysis to reform the parent aglycone, and intra-molecular rearrangement. Three human UDP-glucuronosyltransferases (UGTs) catalyzing the hepatic glucuronidation of carboxylic acid drugs have been identified, UGT1A3, UGT1A9 and a UGT2B7 variant. Within the liver, acyl glucuronides also undergo enzymatic hydrolysis by beta-glucuronidase and esterases which, like the UGTs, are located in the endoplasmic reticulum. In addition, the liver also transports acyl glucuronides between the sinusoidal circulation and bile. Due to their polarity, membrane transport of acyl glucuronides is carrier-mediated, resulting in the establishment of significant concentration gradients between sinusoidal circulation, hepatocyte and bile, in the order of 1:50:5,000 in these compartments, respectively. As a result of exposure to high acyl glucuronide concentrations, the liver is a major target of protein adduct formation. Dipeptidylpeptidase IV, UGTs and tubulin have been identified as intra-hepatic targets of adduct formation by acyl glucuronides. Adduct formation results in altered protein activity and potentially contributes to hepatotoxicity. Hepatic protein adducts are also immunogenic and may cause immune mediated cytotoxicity. Both intra- and extra-hepatic exposure to acyl glucuronides depends not only on the efficiency of glucuronidation and hydrolysis by the liver, but also on the efficiency of the hepatic membrane transport systems. Thus, changes in membrane transporter activities, as may occur due to saturation or drug-drug interactions, can significantly affect acyl glucuronide disposition, adduct formation and the disposition of parent aglycone, thereby affecting clinical efficacy and toxicity of acyl glucuronide forming drugs.

Effects of gemfibrozil and clofibric acid on the uptake of taurocholate by isolated rat hepatocytes.

Clinical use of fibrate hypolipidaemic agents has been associated with an increased incidence of hepatobiliary dysfunction including increased bile lithogenicity, gallstone formation, and cholestasis. The hepatic transport of bile acids plays an important role in bile formation and flow, and interference with the hepatocellular transport of bile acids may result in hepatobiliary dysfunction. The aim of this study was to investigate the effects of gemfibrozil and clofibric acid on the uptake of taurocholate by rat isolated hepatocytes. In control hepatocyte preparations (N = 5) at 37 degrees, the uptake of taurocholate was described by saturable Michaelis-Menten kinetics with a mean (+/-SD) Km of 44.1 +/- 10.2 microM and Vmax of 62.0 +/- 23.0 nmol/10(6) cells/min. In the presence of 200 microM clofibric acid, there was no significant change in the kinetics of taurocholate uptake. However, in the presence of 200 microM gemfibrozil there was a statistically significant (P < 0.05) decrease in the Vmax of taurocholate uptake (32.0 +/- 18.2 nmol/10(6) cells/min, N = 5) and no change (P > 0.05) in Km (48.5 +/- 29.5 microM, N = 5). Gemfibrozil behaved as a non-competitive inhibitor of taurocholate uptake, with a Ki of 144 microM, which is approximately 50 times higher than the unbound gemfibrozil concentrations achieved clinically in humans. Thus, gemfibrozil and clofibric acid did not appear to directly alter the hepatic uptake of taurocholate at clinically relevant concentrations.

The stereospecific incorporation of fenoprofen into rat hepatocyte and adipocyte triacylglycerols.

The formation of triacylglycerols containing fenoprofen was studied in rat isolated adipocytes and hepatocytes incubated with [3H]glycerol and R or S fenoprofen. In both hepatocytes and adipocytes there was a high-affinity enzymatic process for the synthesis of triacylglycerol containing fenoprofen which was stereospecific for the R enantiomer. The apparent Km values for R fenoprofen were 1.0 microM in adipocytes and 2.8 microM in hepatocytes. These results are consistent with the proposed stereospecific formation of R-2-arylpropionyl-CoA thioesters resulting in the stereospecific formation of R-tri-acylglycerol at clinically relevant unbound fenoprofen concentrations. In isolated hepatocytes, but not adipocytes, a second low-affinity enzymatic process for the synthesis of triacylglycerol containing fenoprofen was also observed. However, this process (Km = 3780 microM) occurred at concentrations much higher than those found in man with usual doses.

In vivo covalent binding of clofibric acid to human plasma proteins and rat liver proteins.

Recent studies have shown that acyl-glucuronide conjugates are chemically reactive electrophilic metabolites that can undergo transacylation reactions resulting in intra-molecular rearrangement, hydrolysis and covalent binding of aglycone to albumin both in vitro and in vivo. The hypolipidaemic agent clofibrate is eliminated almost entirely as clofibric acid glucuronide in humans and rats. The formation of clofibric acid-protein adducts was investigated in 14 patients receiving 0.5-2.0 g/day of clofibrate for hypercholesterolaemia, and in liver homogenates from 20 rats administered 280 mg/kg/day of clofibric acid for up to 21 days. Total clofibric acid concentrations in the patients ranged from 0 to 114 mg/L. Covalently bound clofibric acid-protein adducts were detected in all patients, even in one subject in whom there was no measurable plasma clofibric acid. Concentrations ranged from 2.2 to 53.4 ng/mg protein and, in eight patients receiving 1.0 g/day of clofibrate, were correlated (P less than 0.05) with renal function as assessed by creatinine clearance. Clofibric acid-protein adducts were also present in rat liver homogenates, and increased with increasing duration of treatment (P less than 0.0001), from a mean (SE) of 10.1 (0.7) to 32.3 (1.6) ng/mg protein. The covalent binding of drugs to tissue macromolecules has traditionally been associated with toxicity. Further research is required to elucidate the role of acyl-glucuronide conjugates in the formation of drug-protein adducts and their biological consequences.

Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography-mass spectrometry.

A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography-mass spectrometry (LC-MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC-MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were

Optimisation of the comet genotoxicity assay in freshly isolated murine hepatocytes: detection of strong in vitro DNA damaging properties for styrene.

While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a "scraping" buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5-10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.

A new simple diagnostic assay for the identification of the major CYP2D6 genotypes by DNA sequencing analysis.

AIM: To establish a method suitable for diagnostic genotyping of CYP2D6 alleles by DNA sequencing. METHODS: Initial PCR reactions were performed to specifically amplify exons 3, 4, 5 and 6 of the CYP2D6 gene using primers previously published. New primers were used to identify *2, *3, *4, *6, *7, *8, *9 and *41 in 2 sequencing reactions. Additional primers were designed for reverse sequencing in samples with 1 or 3 b.p. deletions. Previously published assays were used to detect *5, *10 and *16 alleles to complete genotype assignment. RESULTS: We reliably detected the nonfunctional alleles, *3, *4, *6, *7 and *8, which are associated with the poor metabolizer phenotype, and 2 important alleles associated with decreased enzyme activity, *9 and *41. Observed allele frequencies were comparable to those found previously in Caucasian populations. CONCLUSION: CYP2D6 genotype has been shown in previous clinical studies to be a good predictor of CYP2D6 phenotype and, therefore, related to therapeutic response and the risk of drug toxicity. This genotyping method is simple and reliable, and, therefore, can be routinely performed on an isolated patient sample, providing a relatively quick turnaround time needed for clinical practice. In addition, the simultaneous drawing of blood with the commencement of drug therapy will allow dosage adjustment on the basis of the CYP2D6 genotype to reduce the risk of adverse drug reactions.

High-performance liquid chromatography quantitation of plasma lamotrigine concentrations: application measuring trough concentrations in patients with epilepsy.

Lamotrigine is a phenyltriazine anticonvulsant recently approved for clinical use. A high-performance liquid chromatographic (HPLC) method was developed using a silica column (5 microm) with an aqueous methanol mobile phase consisting of 94% methanol, 5.92% water, and 0.08% NH4H2PO4 adjusted to a final apparent pH of 4.0 and pumped at a flow rate of 1.0 ml/minute. Ultraviolet detection was carried out at a wavelength of 280 nm, and plasma samples were prepared for HPLC analysis by extraction into ethyl acetate after basification. Retention times for lamotrigine and its internal standard (BWA725C) were 10.3 and 11.2 minutes, respectively, and there was no chromatographic interference from other commonly coadministered anticonvulsants. Calibration curves were linear over a concentration range of 0.5 to 30 mg/l, with intra-assay and interassay coefficients of variation less than 8%. Assessment of assay performance in an international quality assurance program showed an average bias of 0.3% compared with the consensus mean. A review of 52 patient specimens showed that, if patients were grouped according to coadministered anticonvulsants, a significant correlation between lamotrigine dosage and concentration was evident in those coadministered valproate (in the absence of metabolic inducers) and in those coadministered a combination of valproate and inducers, but not in patients coadministered inducers alone. Mean (SD) trough concentrations were 9.2 (5.2), 2.8 (1.3), and 3.8 (2.8) mg/l in the valproate, inducer, and combination groups, respectively.

Unbound plasma phenytoin concentrations measured using enzyme immunoassay technique on the cobas MIRA analyser--in vivo effect of valproic acid.

This communication describes a modification of the total plasma phenytoin enzyme immunoassay technique (EMIT) run on the Cobas MIRA analyser that allows quantitation of unbound phenytoin concentrations in human plasma for routine therapeutic drug monitoring (TDM) purposes. An application of this method is also presented to consider the previously described protein binding drug interaction with concomitantly administered valproic acid in patients with epilepsy. The coefficients of variation for the unbound phenytoin assay ranged from 7.5 to 9.6% and the assay had a reproducibility and accuracy similar to the total phenytoin assay, acceptable for routine TDM. Phenytoin protein binding was linear over a range of total plasma concentrations of 3-65 mg/L. Patients also receiving valproic acid (nine patients, 105 specimens) had a significantly (p less than 0.0001) greater mean +/- SD unbound phenytoin fraction (13.3 +/- 3.1%) compared to nine patients (110 specimens) not receiving valproic acid (8.3 +/- 1.6%). There was also a significant correlation (p less than 0.001) between plasma valproic acid concentration and unbound phenytoin fraction, which resulted in greater intrasubject variability in phenytoin protein binding.

Assessment of interlaboratory performance in the provision of perhexiline therapeutic drug monitoring services in Australia.

Perhexiline is a prophylactic antianginal agent particularly useful in patients whose angina is poorly controlled or refractory to conventional drug regimens. Although perhexiline can cause serious hepatic and neurological toxicity, maintaining trough plasma concentrations between 0.15-0.60 mg/L minimizes the risk of toxicity while providing relief of angina symptoms in a majority of patients. All pathology laboratories are required to participate in interlaboratory proficiency testing (PT) programs. The authors therefore initiated a monthly PT program to assess the performance of Australian laboratories measuring perhexiline (n = 8). PT specimens included perhexiline-spiked drug-free human plasma and pooled plasma from patients administered perhexiline. The performance of 8 Australian laboratories participating in the program was examined over a 30-month period. The mean relative standard deviation of the group was 18.2%. All centers performed well with respect to accuracy, achieving mean percentage bias within +/-8% of target perhexiline concentrations. The usefulness of the PT program was highlighted by the identification of two laboratories with an unacceptable degree of variability (up to 30% of results varied more than +/-55% from the target concentration), and the identification of potential analytical problems with the use of perhexiline metabolite concentrations for determining patients' hydroxylator status. Continued and improved use of PT by pathology laboratories is essential to ensuring the safe and effective clinical use of perhexiline.

In vivo perturbation of rat hepatocyte canalicular membrane function by diclofenac.

Clinical use of diclofenac is associated with a small but significant incidence of hepatotoxicity. It has been reported that in vivo diclofenac treatment results in decreased activity of the extracellular canalicular membrane protein dipeptidylpeptidase IV in rats as a consequence of protein adduct formation by its electrophilic metabolite diclofenac acyl glucuronide. The present study has investigated the effects of in vivo diclofenac treatment (15 mg/kg/day for 7 days) on the activity of an another four rat extracellular canalicular membrane proteins. Animals administered diclofenac (n = 6) had 47.9, 60.4, and 51.6% lower (p < 0.05) canalicular activities of gamma-glutamyltransferase, Mg(2+)-ATPase, and leucine aminopeptidase, respectively, compared with controls (n = 6), but there was no difference in alkaline phosphatase activity. In general, protein adduct formation by acyl glucuronides has been associated with decreased protein function, and the lower canalicular enzyme activities in diclofenac-treated rats may suggest that gamma-glutamyltransferase, Mg(2+)-ATPase, and leucine aminopeptidase are also targets of adduct formation by acyl glucuronide metabolites of diclofenac. However, intracellular redistribution and/or decreased synthesis of these enzymes would also be consistent with our results. The ability of diclofenac acyl glucuronide (200 microg/ml) to form covalently bound adducts with gamma-glutamyltransferase (10 mg/ml) was demonstrated following in vitro incubations (16 h, pH 7.4, and 37 degrees C) in which 20.7 +/- 2.1 ng of diclofenac were covalently bound per milligram of protein. In these in vitro studies, the low concentration of protein adducts formed was not associated with any significant change in gamma-glutamyltransferase activity.

Reactivity of gemfibrozil 1-o-beta-acyl glucuronide. Pharmacokinetics of covalently bound gemfibrozil-protein adducts in rats.

Acyl glucuronides are electrophilic metabolites that are readily hydrolyzed, undergo intramolecular rearrangement, and mediate the covalent binding of many acidic drugs to endogenous proteins. Gemfibrozil is extensively metabolized to gemfibrozil acyl glucuronide in humans and rats. The aims of this study were to demonstrate the reactivity of gemfibrozil glucuronide, determine whether gemfibrozil formed covalently bound protein adducts in vivo, describe the pharmacokinetics of adduct formation, and examine the role of gemfibrozil glucuronide in adduct formation. Rats were administered 150 mg/kg gemfibrozil daily for up to 37 days and killed 1, 2, 5, 10, 19, and 37 days after commencement of dosing, and 1, 2, 3, 8, 17, and 30 days after cessation of dosing. Plasma, liver, kidney, and heart were examined for adduct formation. Plasma was quantitatively the most important site for formation of gemfibrozil-protein adducts with mean (SE) steady-state concentrations of 31.40 (2.40) ng/mg protein attained by approximately the 10th day of dosing. Adduct half-life in plasma was 3.1 days, consistent with the elimination half-life of albumin. Mean (SE) kidney, liver, and heart steady-state adduct concentrations were 2.13 (0.11), 0.89 (0.35), and 0.95 (0.07) ng/mg protein, respectively. The rate of gemfibrozil-protein adduct accumulation seemed greatest in liver, but was similar in kidney and plasma, with approximately 2x, 16x, and 30x accumulation, respectively, over the dosing interval. In all tissues, adduct half-lives were significantly greater than those of the noncovalently bound gemfibrozil or gemfibrozil glucuronide.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis, characterisation and direct high-performance liquid chromatographic analysis of gemfibrozil 1-O-beta-acylglucuronide.

Gemfibrozil 1-O-beta-acylglucuronide was purified from the urine of a volunteer administered gemfibrozil, and an isocratic reversed-phase HPLC method was developed for its direct measurement. Quantitation of gemfibrozil and gemfibrozil 1-O-beta-acylglucuronide was carried out from plasma, following extraction from acidified specimens into ethyl acetate, on a 5-microns CN reversed-phase column with a mobile phase (pH 3.5) containing acetonitrile, tetrabutylammonium sulphate and distilled water, using fluorescence detection at 284 nm excitation and 316 nm emission. Calibration curves were linear for both compounds over a concentration range of 0.1 to 40 mg/l, with intra-assay coefficients of variation < 5% at concentrations of 20.0, 2.0 and 0.2 mg/l, and inter-assay coefficients of variation < 10%. No degradation of gemfibrozil 1-O-beta-acylglucuronide was detected as a result of the analytical procedure. However, a preliminary application of the method indicates that gemfibrozil acylglucuronide is chemically unstable undergoing intra-molecular rearrangement and hydrolysis under physiological conditions.

High-performance liquid chromatographic quantitation of triacylglycerols containing fenoprofen from biological samples.

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the quantitation of radiolabelled triacylglycerols containing fenoprofen, synthesized from [3H]glycerol by isolated hepatocytes and adipocytes. The assay consists of extracting the lipids into diethyl ether, separating triacylglycerols from polar endogenous lipids using silica Sep-Pak cartridges and quantitating endogenous triacylglycerols and triacylglycerols containing fenoprofen by HPLC resolution and scintillation counting. HPLC separation is achieved in less than 10 min. Using [14C]tripalmitin as internal standard the assay has a linear relationship between added triacylglycerol and measured endogenous triacylglycerols and triacylglycerols containing fenoprofen with regression coefficients of 0.997 and 0.998, respectively.

Alterations in prednisolone disposition as a result of time of administration, gender and dose.

The disposition of total and free prednisolone has been studied in four male and four female volunteers, each of whom received an intravenous dose of 0.075 mg/kg (low) and 1.5 mg/kg (high) of prednisolone at both 06.00 h and 18.00 h. For the low dose, free prednisolone clearance was 14% lower (P = 0.012) and time-averaged prednisolone free fraction was 22% higher (P less than 0.001) in the morning, there being no circadian difference in total prednisolone clearance. There was no circadian differences in prednisolone disposition at the high dose. These findings are consistent with a mechanism in which cortisol causes a simultaneous competitive inhibition of prednisolone clearance and plasma protein binding at low, but not at high prednisolone doses. Prednisolone clearance was higher in female than in male subjects, the mean increase being 18% (P = 0.022) for total prednisolone and 21% (P = 0.036) for free prednisolone. Mean total prednisolone clearance and steady-state distribution volume were two-fold higher at the high vs the low dose (P less than 0.001), but free prednisolone clearance showed a dose dependent decrease of 11% (P = 0.019). There was no change in free prednisolone steady-state distribution volume.

Interaction of human serum albumin with the electrophilic metabolite 1-O-gemfibrozil-beta-D-glucuronide.

Acyl glucuronides are electrophilic metabolites that are readily hydrolyzed, undergo intramolecular rearrangement, and bind covalently to endogenous proteins. Gemfibrozil is a fibrate lipid-lowering agent that is extensively metabolized to an acyl glucuronide conjugate in humans. The aims of this study were to examine the interactions of 1-O-gemfibrozil-beta-D-glucuronide with human serum albumin. The degradation of 1-O-gemfibrozil-beta-D-glucuronide (approximately 200 microM) was examined in vitro during incubations at 37 degrees C with phosphate buffer (pH 7.4 or 9.0), solutions of human serum albumin (pH 7.4), or fresh human plasma (pH 7.4). The effects of diazepam, oxyphenbutazone, and gemfibrozil on the degradation of 1-O-gemfibrozil-beta-D-glucuronide, and its reversible binding to albumin were also studied. A pilot in vivo study was performed on two patient volunteers administered 1 g/day p.o. gemfibrozil. 1-O-Gemfibrozil-beta-D-glucuronide was unstable, with degradation half-lives in buffer of 4.1 hr and 44 hr at pH 9.0 and 7.4, respectively; and 8.5 hr and 5.5 hr in pH 7.4 solutions of human serum albumin or fresh plasma, respectively. Degradation was dependent on pH and the presence of albumin, which seemed to accelerate the intramolecular rearrangement and hydrolysis of the conjugate. 1-O-Gemfibrozil-beta-D-glucuronide was highly reversibly bound to albumin, with a mean unbound fraction of 0.028, and its degradation seemed to be related to the degree of reversible binding. Hydrolysis and covalent binding were associated with the site II binding domain on albumin, because only diazepam inhibited these reactions. However, intramolecular rearrangement was increased when binding to the site I domain was inhibited. Covalent binding was also detected in vivo to human plasma proteins. The half-life of the gemfibrozil-protein adducts was 2.5-3 days. Albumin plays an important role in the disposition of acyl glucuronides by acting as: i) a transporter protein; ii) a potential catalyst for their degradation and, therefore, clearance; and iii) a target for covalent adduct formation.

High-performance liquid chromatographic determination of sotalol in plasma. I. Application to the disposition of sotalol enantiomers in humans.

Two high-performance liquid chromatographic analytical methods have been developed for the measurement of dl-sotalol or d-sotalol and l-sotalol in plasma, using dl-atenolol as internal standard. Quantitation of dl-sotalol was carried out, following solid-phase extraction, on a 5-microns C18 reversed-phase column, with a mobile phase containing acetonitrile, ion-pairing reagent and distilled water, using ultraviolet detection at 235 nm. Quantitation of d-sotalol and l-sotalol was based on derivatisation with the chiral agent S-(-)-alpha-methylbenzyl isocyanate, followed by chromatographic separation on a 3-microns C18 reversed-phase column, with a mobile phase containing methanol, glacial acetic acid and distilled water, with fluorimetric detection at 220 nm excitation and 300 nm emission. A preliminary application of the latter method suggests that the disposition of sotalol in humans is not enantioselective.

High-performance liquid chromatography determination of clonazepam in plasma using solid-phase extraction.

The use of high-performance liquid chromatography for therapeutic drug monitoring of clonazepam has previously been limited by low sensitivity and labor-intensive liquid-liquid extractions. The present method was developed employing a rapid solid-phase extraction, thus minimising sample workup and providing analytical sensitivity down to 2 micrograms/L using 1 ml of plasma. Plasma samples were loaded onto C18 solid-phase extraction columns, and clonazepam and its internal standard (methyl-clonazepam) were eluted with methanol, dried, and reconstituted in 130 microliters of mobile phase. Chromatographic separation was achieved using a 3-microns RP18 column at 40 degrees C and a mobile phase of 32% acetonitrile and 0.5% glacial acetic acid in distilled water at 0.5 ml/min. Detection was carried out using ultraviolet absorbance at 306 nm. Retention times for clonazepam and methyl-clonazepam were approximately 7 and 12 min respectively. Standard curves were linear over a range of 5-200 micrograms/L with intraassay coefficients of variation of 1.2 and 4.8% at 200 and 5 micrograms/L, respectively. Plasma concentrations measured in patient samples were not statistically different from those obtained using an established gas chromatographic method, and quality control specimens from the Heathcontrol EQA Scheme were consistently within +/- 1.2 SD of the group means. There was no chromatographic interference from other benzodiazepines or other drugs used for the treatment of epilepsy.