Hydrolysis and Enantiodiscrimination of (R)- and (S)-Oxazepam Hemisuccinate by Methylated ß-Cyclodextrins: An NMR Investigation.

Partially and exhaustively methylated ß-cyclodextrins [(2-methyl)-ß-CD (MCD), heptakis-(2,6-di-O-methyl)-ß-CD (DIMEB), and heptakis-(2,3,6-tri-O-methyl)-ß-CD (TRIMEB)] have been compared in the hydrolysis and enantiodiscrimination of benzodiazepine derivative (R)- or (S)-oxazepam hemisuccinate (OXEMIS), using nuclear magnetic resonance (NMR) spectroscopy as an investigation tool. After 6 h, MCD induced an 11% hydrolysis of OXEMIS, remarkably lower in comparison with underivatized ß-CD (48%), whereas no hydrolysis was detected in the presence of DIMEB or TRIMEB after 24 h. DIMEB showed greater ability to differentiate OXEMIS enantiomers in comparison to TRIMEB, by contrast MCD did not produce any splitting of racemic OXEMIS resonances. Both enantiomers of OXEMIS underwent deep inclusion of their phenyl pendant into cyclodextrins cavities from their wider rims, but tighter complexes were formed by DIMEB with respect to TRIMEB.

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.

Evaluation of abalone ß-glucuronidase substitution in current urine hydrolysis procedures.

This study examined the potential of abalone ß-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia ß-glucuronidase and Escherichia coli ß-glucuronidase. Abalone ß-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia ß-glucuronidase. JWH 018 N-(5-hydroxypentyl) ß-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone ß-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli ß-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone ß-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone ß-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone ß-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids.

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.

Quantitation of benzodiazepines in urine, serum, plasma, and meconium by LC-MS-MS.

A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within +/- 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within +/- 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.

The concentration of oxazepam and oxazepam glucuronide in oral fluid, blood and serum after controlled administration of 15 and 30 mg oxazepam.

AIMS: To measure and compare the concentration-time profiles of oxazepam and oxazepam glucuronide in blood, serum and oral fluid within the scope of roadside testing. METHODS: Biological samples were collected from eight male subjects after ingestion of 15 or 30 mg oxazepam on separate dosing occasions with an interval of 7 days. The concentration-time profiles of oxazepam and oxazepam glucuronide were fitted by using a one-compartment model. RESULTS: For oxazepam and oxazepam glucuronide, the mean oral fluid/blood ratios were 0.05 (range 0.04-0.07) and 0.004 (range 0.002-0.006), respectively. The concentration-time profiles in oral fluid paralleled those in blood. CONCLUSION: After oral administration of therapeutic doses of oxazepam, concentrations in oral fluid are very much lower than those in blood, and those of oxazepam glucuronide are much lower than those of the parent compound. Nevertheless, assay of oral fluid for oxazepam can be used to detect recent ingestion of the drug in drivers suspected of impaired driving performance.

Chromatographic analysis of allosteric effects between ibuprofen and benzodiazepines on human serum albumin.

The effects of (R)- and (S)-ibuprofen on the binding of benzodiazepines to human serum albumin (HSA) were examined by biointeraction chromatography. The displacement of benzodiazepines from HSA by (R)- and (S)-ibuprofen was found to involve negative allosteric interactions (or possible direct competition) for most (R)-benzodiazepines. However, (S)-benzodiazepines gave positive or negative allosteric effects and direct competition when displaced by (R)- or (S)-ibuprofen. Association equilibrium constants and coupling constants measured for these effects indicated that they involved two classes of ibuprofen binding regions (i.e., low- and high-affinity sites). Based on these results, a model was proposed to explain the binding of benzodiazepines to HSA and their interactions with ibuprofen. This model gave good agreement with previous reports examining the binding of benzodiazepines to HSA.

New series of morpholine and 1,4-oxazepane derivatives as dopamine D4 receptor ligands: synthesis and 3D-QSAR model.

Since the identification of the dopamine D(4) receptor subtype and speculations about its possible involvement in schizophrenia, much work has been put into development of selective D(4) ligands. These selective ligands may be effective antipsychotics without extrapyramidal side effects. This work describes the synthesis of a new series of 2,4-disubstituted morpholines and 2,4-disubstituted 1,4-oxazepanes with selectivity for the dopamine D(4) receptor. A 3D-QSAR analysis using the GRID/GOLPE methodology was performed with the purpose to get a better understanding of the relationship between chemical structure and biological activity. Inspection of the coefficient plots allowed us to identify that regions which are important for affinity are situated around the two benzene ring systems, a p-chlorobenzyl group, and the aliphatic amine belonging to the morpholine or 1,4-oxazepane system. In addition, the size of the morpholine or 1,4-oxazepane ring seems to be important for affinity.

(S)oxazepam glucuronidation is inhibited by ketoprofen and other substrates of UGT2B7.

1,4-Benzodiazepine anxiolytics such as diazepam and halazepam are converted in vivo to oxazepam, an active metabolite with a hydroxyl group at the asymmetric C3 position. D-glucuronic acid couples with the C3 hydroxyl group of oxazepam to form pharmacologically inactive diastereomeric glucuronide conjugates. Conjugation with glucuronic acid is catalysed by the microsomal UDP-glucuronosyltransferase (UGT) enzyme system, which includes an undetermined number of isozymes. Although 1,4-benzodiazepines are ultimately cleared as oxazepam glucuronide, little is known about the particular UGT isozyme(s) responsible for the conjugation at the C3 position of these molecules. Microsomal preparations from three human livers were used to study the glucuronidation of (R,S)oxazepam in vitro. The predominant formation of the S- over the R-glucuronide was reflected by the kinetic parameters: For (S)oxazepam glucuronide, the constants were Km = 0.18 +/- 0.02 mM and Vmax = 202.6 +/- 25.0 nmol min-1 per mg protein; for (R)oxazepam glucuronide, they were Km = 0.22 +/- 0.02 mM, Vmax = 55.4 +/- 9.5 nmol min-1 per mg protein. Inhibition studies suggest that the two diastereomeric glucuronidations are catalysed by different UGT isozymes. That is, there was competitive inhibition of (S)oxazepam glucuronidation by non-steroidal anti-inflammatory drugs (NSAIDs), including ketoprofen while (R)oxazepam glucuronidation was not equally inhibited by these compounds. The order of potency of inhibitors of (S)oxazepam glucuronidation in this study was the same as the rank order of substrates conjugated by UGT2B7; hyodeoxycholic acid, estriol, (S)naproxen, ketoprofen, ibuprofen, fenoprofen, clofibric acid, and morphine (in descending order). The inhibition profile of (S)oxazepam glucuronidation suggests that UGT2B7 is the catalysing enzyme.