Deposition of diazepam and its metabolites in hair following a single dose of diazepam.

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.

Direct determination of diazepam and its glucuronide metabolites in human whole blood by µElution solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A µElution solid-phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam and temazepam glucuronide in human whole blood is presented. 200 µL of whole blood samples were loaded onto a Waters Oasis HLB 96-well µElution SPE plate using 75 µL of methanol as the elution solvent, and the eluents were injected into an Eclipse XDB C18 column. No hydrolysis, solvent transfer, evaporation or reconstitution was involved in the sample preparation procedures. Tandem mass spectrometric detection with Turbo Ion Spray was conducted via multiple reaction monitoring (MRM) under positive ionization mode. The method was validated and proved to be accurate (accuracy within 93-108%), precise (intra-day RSD<9.9% and inter-day RSD<7.2%) and sensitive with limits of detection (LOD) in the range of 0.05-0.25 ng/mL for all the compounds. Extraction recoveries were in the range of 31-80% for all the analytes. This method demonstrated to be reproducible and reliable. The applicability of the method was demonstrated by analysis of several forensic cases involving diazepam and its metabolites.

Strain differences in diazepam metabolism at its three metabolic sites in sprague-dawley, brown norway, dark agouti, and wistar strain rats.

Knowledge of strain differences in drug metabolism is important for the selection of animals for pharmacokinetic, pharmacodynamic, and toxicological studies. Hepatic microsomes from Sprague-Dawley (SD) and Brown Norway (BN) rats had 300-fold higher diazepam p-hydroxylation activity than Dark Agouti (DA) and Wistar (W) rats at a low diazepam concentration (3 microM). Kinetic studies indicated that diazepam p-hydroxylation in SD and BN rats proceeded with lower K(m) and higher V(max) values than it did in DA and W rats. However, the expression levels of cytochrome P450 CYP2D1, the reported enzyme for diazepam p-hydroxylation, did not cosegregate with the activity. These results suggest the presence of a new high-affinity diazepam p-hydroxylation enzyme other than CYP2D1 in SD and BN rats. DA rats showed 3- and 2-fold higher diazepam 3-hydroxylation and N-desmethylation activities, respectively, than the other rat strains. In agreement with this, DA rat liver microsomes had a higher expression of CYP3A2, which is responsible for diazepam 3-hydroxylation and partly responsible for N-desmethylation. Values of CL(int) (V(max)/K(m)) indicated that p-hydroxy-diazepam is the major metabolite in SD and BN rats, whereas 3-hydroxy-diazepam is the major metabolite in DA and W rats. The sum of the CL(int) in each strain was in the order of DA > SD = BN >> W. Strain differences in the pharmacodynamics of diazepam between SD and DA rats may be due to these differences in diazepam metabolism. We found that both the rate of elimination of diazepam and the major metabolic pathways in diazepam metabolism differed among the different rat strains due to polymorphic expression of the two enzymes involved in diazepam metabolism.

Highly stereoselective acid-catalyzed homonucleophilic substitution reactions.

Enantiomeric 3-O-methyltemazepam and 3-O-ethyltemazepam were highly stereoselectively substituted by the 3-methoxy group of methanol in acidic anhydrous methanol and by the 3-ethoxy group of ethanol in acidic anhydrous ethanol, respectively. The stereoselectivity of the homonucleophilic substitution reactions was determined by circular dichroism spectropolarimetry and gas chromatography-mass spectrometry. In anhydrous solutions containing 0.5 M D2SO4 at 50 degrees C, for example, the stereoselectivity was approximately 63:1 for enantiomeric 3-O-methyltemazepam in CD3OD and approximately 94:1 for enantiomeric 3-O-ethyltemazepam in C2D5OD. The high stereoselectivity at C3 position was primarily due to the presence of a methyl group at N1 position.

Achiral and chiral analysis of camazepam and metabolites by packed-column supercritical fluid chromatography.

Supercritical fluid chromatography, using carbon dioxide as the mobile phase and ethanol as a modifier, has been applied to the analysis of products formed in rat liver microsomal metabolism of racemic camazepam, a hypnotic/anxiolytic drug in clinical use. An achiral (amino) column and a chiral (Chiralcel OD-H) column were used. The results suggest that achiral and chiral packed-column supercritical fluid chromatography gives a shorter analysis time and higher selectivity and efficiency than achiral and chiral stationary-phase high-performance liquid chromatography in the analysis of camazepam and its derivatives.

Acid-catalyzed stereoselective heteronucleophilic substitution and racemization of 3-O-methyloxazepam and 3-O-ethyloxazepam.

Enantiomers of 3-O-methyloxazepam (MeOX) and 3-O-ethyloxazepam (EtOX) were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Reaction kinetics and deuterium isotope effects of acid-catalyzed racemization of enantiomeric MeOX in ethanol and enantiomeric EtOX in methanol were studied by spectropolarimetry. The acid-catalyzed heteronucleophilic substitution reactions of racemic MeOX in ethanol and racemic EtOX in methanol were studied by reversed-phase HPLC. Thermodynamic parameters involved in the reactions were obtained by temperature-dependent reaction rates. The effects of solvent's dielectric constant on the heteronucleophilic substitution reactions were also determined. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer, derived from a 1,4-benzodiazepine either in M (minus) or P (plus) conformation, is proposed to be an intermediate and responsible for the acid-catalyzed stereoselective nucleophilic substitution and the resulting racemization.

Stereoselective homonucleophilic substitution of 3-O-methyl and 3-O-ethyloxazepam enantiomers by chiral stationary phase high-performance liquid chromatography.

Enantiomers of 3-O-methyloxazepam and 3-O-ethyloxazepam were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Temperature-dependent and acid-catalysed racemization of 3-O-methyloxazepam enantiomers in methanol and 3-O-ethyloxazepam enantiomers in ethanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of reaction product were determined by CSP-HPLC. Thermodynamic parameters in the formation of the activated complex (Eact, delta H++, delta S++ and delta G++) were consistent with those determined by a spectropolarimetric method. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer is proposed to be an intermediate and responsible for the acid-catalysed stereoselective homonucleophilic substitution and the resulting racemization.

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography.

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with beta-glucuronidase. Separation is achieved using a C8 reversed-phase column with a methanol-water-phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 micrograms/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.

Hypnotic activity and effects on performance of lormetazepam and camazepam--analogues of temazepam.

1 The effects of lormetazepam and camazepam on sleep electroencephalography, visuo-motor coordination, digit symbol substitution and subjective assessments of mood and sleep quality were compared with placebo in six young adult males (18-27 years). The study was double blind. 2 Over the dose range 0.5, 1.0 and 2.0 mg, lormetazepam increased total sleep time (P less than 0.05), reduced wakefulness (P less than 0.05) and drowsy sleep (linear effect P less than 0.05). With 2.0 mg there were increases in stage 3 (P less than 0.05) and reduction in rapid eye movement sleep (P less than 0.01). Overnight ingestion of 2.0 mg, was followed by impaired visuo-motor coordination and fewer substitutions with the digit symbol test. 3 The hypnotic effect of 10-20 mg camazepam was limited to reduced awake activity (P less than 0.05), and with 20 mg there were increased substitutions on the digit symbol test. After 40 mg overnight stage 4 sleep was reduced (P less than 0.001) and performance at the digit symbol test was impaired (P less than 0.05 at 9.75 h). Morning ingestion of 20 mg camazepam did not alter performance, and the subjects assessed themselves to be more relaxed. 4 Lormetazepam is not specially indicated for those involved in skilled activity, but may prove useful for patients with insomnia resistant to other drugs. Camazepam would appear to be a promising anxiolytic with minimal effects on performance.