Oxazepam Glucuronide Research Articles

B-271 High Sensitivity Homogeneous Enzyme Immunoassay for Benzodiazepines and Metabolites

Abstract Background Benzodiazepines are widely prescribed for treating various conditions such as anxiety, insomnia, and seizures due to their sedative and anxiolytic properties. Despite their therapeutic benefits, concerns persist regarding their potential for misuse, dependence, and addiction. Urine drug screening immunoassays serve as an important tool in monitoring benzodiazepine usage, ensuring adherence to prescribed regimens, identifying misuse or abuse, and facilitating necessary medical interventions. However, limitations of benzodiazepine immunoassays are well-documented in literature, with issues surrounding their lack of sensitivity to glucuronidated and 7-amino metabolites, inability to detect newer benzodiazepines, and poor cross-reactivity with different benzodiazepines. ARK Diagnostics has developed a highly sensitive homogeneous enzyme immunoassay capable of detecting both classic and designer benzodiazepines, in addition to their glucuronide and 7-amino metabolites in human urine at a cutoff concentration 200 ng/mL, without the need for glucuronidase or sample pretreatment. Methods The ARKTM Benzodiazepine Plus Assay is a liquid-stable homogenous enzyme immunoassay consisting of two reagents. The assay uses temazepam as the 200 ng/mL cutoff calibrator. The performance characteristics of this assay, including precision, spiked recovery, specificity, and method comparison to LC-MS/MS, were evaluated on the Beckman Coulter AU680 automated clinical analyzer. Results The ARKTM Benzodiazepine Plus demonstrated acceptable precision, with no overlap between cutoff (200 ng/mL) and ±25% control levels (150 ng/mL and 250 ng/mL) in a histogram overlap analysis, and ≤3.6% CV in semi-quantitative mode. Spiked temazepam samples spanning the semi-quantitative assay range of 1000 ng/mL were recovered between 95.0% and 103.7% of the spiked levels. Thirty-eight benzodiazepines produced cross-reactivities >80%, including the following benzodiazepines and metabolites: lorazepam, clonazepam, diazepam, alprazolam, temazepam, oxazepam glucuronide, lorazepam glucuronide, temazepam glucuronide, 7-aminoclonazepam, and 7-aminoflunitrazepam. In method comparison studies, a total of 103 urine samples containing benzodiazepines with LC-MS/MS values ranging from 1.3 ng/mL to 3257.6 ng/mL were tested with the ARKTM Benzodiazepine Plus Assay. Eighty samples tested positive with values ≥200 ng/mL and 23 samples tested negative with values <200 ng/mL by the ARKTM Benzodiazepine Plus assay. Of the 23 samples that tested negative, 21 had LC-MS/MS values <200 ng/mL and 2 samples were identified as midazolam samples with metabolite a-OH-midazolam levels >200 ng/mL. Conclusions The ARKTM Benzodiazepine Plus Assay enables the sensitive, rapid, and reliable measurement of benzodiazepines and their metabolites in human urine, without the need for hydrolysis or pretreatment. The assay addresses the present constraints of currently available benzodiazepine immunoassays, including their insufficient sensitivity, limited cross-reactivity to metabolites, and the requirement for glucuronidase pretreatment. The ARKTM Benzodiazepine Plus Assay is applicable to a wide range of clinical chemistry analyzers.

Stability of diazepam’s phase II metabolites in dried blood spots on filter paper

Phase II metabolites play an important role in diazepam-related cases. The study aimed to assess the stability of diazepam’s phase II metabolites in dried blood spots on filter paper. MethodsA piece of filter paper was spotted with 100 µL of whole blood (added 1% sodium fluoride as needed) obtained from participant who received 5 mg diazepam orally, air dried for 2 h at room temperature, and then stored at different conditions. Whole spots were cut at 0.1 cm from the outer edge of blood spots at post-consumption time-points of prior (zero), 5, 16, 35, 61, 120 days and 1, 1.5 years. Analytes were extracted with methanol/water mixture (8:2, v/v) and determined using HPLC-MS/MS. Decomposition rules were analyzed by a statistical software “SPSS”. ResultsTemazepam glucuronide remained stable (0.5–18.6% loss) at 20 ℃ and at 20 ℃ with 1% sodium fluoride for 16 days, while it was unstable after 5 days at 4 ℃ (21.1–26.2% loss) and − 20 ℃ (28.9 - 34.4% loss). After 35 days, temazepam glucuronide concentrations began to fluctuate significantly under all conditions, and an obvious increase (290.4–355.1%) was observed in 1.5 years. Oxazepam glucuronide was always unstable after 5 days, the percentage loss was even 100% when it was stored for 61 days and 1.5 years. ConclusionsDried blood spots on ordinary filter paper are recommended to be stored at 20 ℃ or 20 ℃ with 1% sodium fluoride within 16 days. Samples should be analyzed immediately or stored in sterile and dry media.

Pharmacokinetics of Diazepam and Its Metabolites in Urine of Chinese Participants.

BackgroundUrine is conventionally used as a specimen to document diazepam-related crimes; however, few reports have described the pharmacokinetics of diazepam and its metabolites in urine.ObjectiveThis study aimed to investigate the pharmacokinetics of diazepam and its metabolites, including glucuronide compounds, in the urine of Chinese participants.MethodsA total of 28 volunteers were recruited and each participant ingested 5 mg of diazepam orally. Ten milliliters of urine were collected from each participant at post-consumption timepoints of prior (zero), 1, 2, 4, 8, 12, and 24 h and 2, 3, 6, 12, and 15 days. All samples were extracted by solid-phase extraction and analyzed using high-performance liquid chromatography–tandem mass spectrometry. Diazepam and its main metabolites, except for temazepam, were detected in the urine of volunteers. Pharmacokinetic parameters were analyzed using the pharmacokinetic software DAS according to the non-compartment model.ResultsUrinary diazepam peaked at 2.38 ng/mL (Cmax) and 1.93 h (Tmax). The urinary metabolite nordiazepam peaked at 1.17 ng/mL and 100.21 h; temazepam glucuronide (TG) peaked at 145.61 ng/mL and 41.14 h; and oxazepam glucuronide (OG) peaked at 101.57 ng/mL and 165.86 h. The elimination half-life (t½z) and clearance (CLz/F) for diazepam were 119.58 h and 65.77 L/h, respectively. The t½z of the metabolites nordiazepam, TG, and OG was 310.58 h, 200.17 h, and 536.44 h, respectively. Finally, this study found that both diazepam and its main metabolites in urine were detectable for at least 15 days, although there were individual differences.ConclusionThe results regarding diazepam pharmacokinetics in urine would be of great help in forensic science and drug screening.

Can Cannabinoids Precipitate UGT‐mediated Drug Interactions?

Background: Cannabidiol (CBD) and tetrahydrocannabinol (THC) are cannabinoids used predominately for medicinal and recreational purposes, respectively. Because these cannabinoids are often co-consumed with conventional drugs, there is concern for deleterious cannabinoid-drug interactions. Both cannabinoids and their major circulating metabolites (7-OH CBD or 11-OH THC) are potent inhibitors of CYPs, but their potency to inhibit UGTs has not been investigated. Objective The objective of the study was to predict the potential for CBD and THC to precipitate UGT-mediated drug interactions using an IVIVE approach. The aims were to determine the following for CBD, THC, and their metabolites: 1) fraction unbound in incubations (fu,inc) containing recombinant UGT (rUGTs) and 0.2% BSA and 2) unbound UGT inhibitory potency (IC50,u). These data were used to predict the potential for interactions between each cannabinoid and UGT-metabolized drugs after typical doses used orally or by inhalation. Methods Fraction unbound (fu,inc) of CBD, THC, or metabolite in incubations containing rUGTs and 0.2% BSA was determined by ultracentrifugation. The inhibition extent (at 10 μM) or potency (IC50,u) of CBD, 7-OH CBD, 7-COOH CBD, THC, 11-OH THC, or 11-COOH THC towards the glucuronidation of estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-OH indole (UGT1A6), propofol (UGT1A9), gemfibrozil (UGT2B4), naloxone (UGT2B7), amitriptyline (UGT2B10), oxazepam (UGT2B15), or testosterone (UGT2B17) was determined using rUGTs. Estimated IC50,u and unbound hepatic inlet (oral dose) or systemic plasma (inhaled dose) concentrations were used to predict the potential for interactions between CBD (700 mg) or THC (20 mg) and drugs metabolized by UGT with fm values of 0.25, 0.5, or 0.75. Results fu,inc of CBD, 7-OH CBD, and THC was 0.05, 0.20, and 0.04, respectively. CBD, 7-OH CBD, THC, and 11-OH THC at 10 μM inhibited all UGTs tested (by 30-98%), whereas 7-COOH CBD inhibited only UGT2B7 (by 25%) and 11-COOH THC inhibited only UGT1A1, 1A4, and 1A9 (by 20-35%). IC50,u of CBD, 7-OH CBD, and THC against UGT1A9, 2B4, and 2B7 ranged from 0.002-0.02 μM, 0.6-1.6 μM, and 0.07-0.13 μM, respectively. IC50,u of CBD against UGT1A4, 1A6, 2B10, and 2B15 was < 1.3 μM. A mechanistic static model predicted strong in vivo interactions (AUC ratio > 3.3) between CBD and drugs extensively metabolized (fm = 0.75) by UGT1A9, 2B4, or 2B7. CBD was also predicted to result in moderate or weak interactions (1.3 ≥ AUC ratio ≤ 2.0) with drugs not extensively metabolized by these UGTs (fm = 0.25 or 0.5). In contrast, oral or inhaled THC was not predicted to precipitate UGT-mediated drug interactions (AUC ratio < 1.2). UGT inhibition by 7-OH CBD did not change the predicted potential of CBD-drug interactions. Conclusion This study is the first to determine the UGT inhibition potencies of cannabinoids. CBD was a potent inhibitor of UGT1A9, 2B4, and 2B7 and was predicted to interact with drugs metabolized by these UGTs. Further investigation via dynamic PBPK modeling and simulation and clinical evaluation is warranted to verify these predictions.

Development and Validation of an LC–MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20μg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30min with a genetically modified β-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes metall quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Study on the Pharmacokinetics of Diazepam and Its Metabolites in Blood of Chinese People.

Driving under the influence of diazepam is increasing in China. The pharmacokinetics of diazepam and its metabolites, especially the glucuronide metabolites, are helpful in the identification of diazepam use by drivers. This study aimed to investigate the pharmacokinetics of diazepam and its metabolites (nordazepam, oxazepam, oxazepam glucuronide and temazepam glucuronide) in the blood of Chinese people, and to provide basic data for identifying diazepam use and estimating the time of last diazepam ingestion. A total of 28 participants (14 men, 14 women) were recruited and each person received 5mg diazepam orally. Whole blood was collected at 0h(pre-dose), and1h, 2h, 4h, 8h, 12h, and 24h, and at 2 days, 3 days, 6 days, 12 days, and 15 days post-dose. Analytes of interest were extracted via solid-phase extraction and analyzed by a liquid chromatography tandem mass spectrometry method operated in a positive multiple response monitoring mode. Pharmacokinetic parameters were analyzed by a pharmacokinetic software DAS according tothe non-compartmentmodel. The time of last diazepam use was estimated using the concentration ratios of diazepam to metabolites and metabolites to metabolites from controlled drug administration studies. The respective time of maximum concentration, the maximum concentration and the elimination half-life of diazepam were 1.04 ± 1.00h, 87.37 ± 31.92ng/mL and 129.07 ± 75.00h; of nordazepam were 133.14 ± 109.63h, 3.80 ± 1.75ng/mL, and 229.73 ± 236.83h; of oxazepam were 100.29 ± 87.16h, 1.62 ± 2.64ng/mL, and 382.86 ± 324.58h; of temazepam glucuronide were 44.43 ± 55.41h, 2.08 ± 0.88ng/mL, and 130.53 ± 72.11h; and of oxazepam glucuronide were 66.86 ± 56.33h, 1.10 ± 0.41ng/mL, and 240.66 ± 170.12h. A good correlation model was obtained from the concentration ratio of diazepam to nordazepam and the time of diazepam use, and the prediction errors were less than 20%. This study provides a sensitive method to identify diazepam ingestion by monitoring diazepam and its metabolites including glucuronides, as well as to infer the time following oral consumption.

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 10mg of diazepam. Hair was collected from all volunteers 1month after exposure, and also 2months post-exposure from men and 10months 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 0.5-2.5pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10pg/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 2months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10months 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.

Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid-phase extraction and liquid chromatography with tandem mass spectrometry.

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

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. 200μL of whole blood samples were loaded onto a Waters Oasis HLB 96-well μElution SPE plate using 75μL 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 0.05–0.25ng/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.

Extending the detection window of diazepam by directly analyzing its glucuronide metabolites in human urine using liquid chromatography–tandem mass spectrometry

A method for the simultaneous direct analysis of diazepam oxazepam glucuronide, temazepam glucuronide, oxazepam, nordiazepam, and temazepam in human urine was developed and validated. Urine sample was purified by solid phase extraction (SPE), and the analysis was achieved using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system equipped with an electrospray ionization source (ESI). Multiple reaction monitoring (MRM) mode was used to analyze the target compounds. Extraction recoveries were 65–122% for all the analytes. The method showed acceptable intra–assay and inter–assay precision (both relative standard deviation (RSD)≤11.2%) for quality control (QC) samples. The limits of detections (LODs) were in the range of 0.1–2ng/mL. The present assay was applied to analyze the urine obtained from three volunteers after oral administration of a single dose 5mg of diazepam. The results showed that, the detection periods of oxazepam glucuronide and temazepam glucuronide were much longer than diazepam and other metabolites.

A Novel Reductive Transformation of Oxazepam to Nordiazepam Observed During Enzymatic Hydrolysis

beta-Glucuronidase is an enzyme often employed to de-conjugate beta-glucuronides during urinary drug testing for benzodiazepines. It is commonly accepted that use of beta-glucuronidase is a preferred method of hydrolysis over acid-catalyzed hydrolysis, which is known to induce benzodiazepine degradation and transformation. Literature to date, however, has not reported any cases of benzodiazepine transformation initiated by commercial beta-glucuronidase products. In this study, urine specimens containing either oxazepam or oxazepam glucuronide were incubated with beta-glucuronidase enzymes obtained from Escherichia coli, Helix pomatia, and Patella vulgata under various incubation conditions. After liquid-liquid extraction, the extract was analyzed by both liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry for the presence of benzodiazepines. All three enzyme preparations examined were capable of reducing oxazepam or oxazepam glucuronide into nordiazepam (desmethyldiazepam). Nordiazepam formation was positively correlated with incubation temperature, incubation time, oxazepam concentration, and enzyme concentration. Under all enzymatic hydrolysis conditions investigated, the percentage of nordiazepam formation is < 2.5% relative to the amount of oxazepam present in the system. The findings of this study have both clinical and forensic implications, and it is clear that the detection of nordiazepam in biological samples subjected to testing involving enzyme-catalyzed hydrolysis should be interpreted with care.

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

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. 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. 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. 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.

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.

Urine Benzodiazepine Screening using Roche Online(R) KIMS Immunoassay with -Glucuronidase Hydrolysis and Confirmation by Gas Chromatography-Mass Spectrometry

Performance of the Roche Online KIMS (kinetic interaction of microparticles in solution) benzodiazepine (BZD) immunoassay (IA) with and without beta-glucuronidase treatment was evaluated on a Hitachi Modular automated IA analyzer calibrated using nordiazepam at 100 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences were evaluated. Precision of the assay (percent coefficient of variation (%CV)) with and without addition of the enzyme was less than 6% and 9%, respectively, with linearity (r(2) value of 0.9578 and 0.9746), respectively. Between-run precision of a 125 ng/mL nordiazepam control (n = 287) over 67 days, produced a %CV of 13.6% for the hydrolytic assay. Modification of the BZD assay to include automated hydrolysis of urinary BZD glucuronide conjugates was evaluated using three glucuronidated BZD standards prepared at concentrations ranging from 250 to 10,000 ng/mL. With hydrolysis, temazepam, oxazepam, and lorazepam glucuronides, produced cross-reactivities of 25%, 15%, and 20%, respectively. Without hydrolysis, the glucuronidated BZD standards produced less than 1% cross-reactivity in the assay. The ability of the assay to differentiate between positive and negative samples was evaluated by assaying 20 negative urine samples and serial dilutions of certified drug-free urine fortified with 28 different BZDs. All of the negative and positive urine samples produced the appropriate screening result. Cross-reactivities of 27 different BZDs, calculated as the normalized IA response divided by the BZD concentration that produced a response approximately equivalent to the response of a 100 ng/mL nordiazepam standard and multiplied by 100, ranged from 15% to 149%. Human urine samples (n = 28) that were previously found to contain BZDs by gas chromatography-mass spectrometry (GC-MS) also produced a positive BZD IA result. The IA was challenged with 78 potentially interfering compounds, and none produced a positive BZD response. As a part of the validation, a large number of human urine samples (29,500) were assayed using the modified Online BZD IA method to evaluate the performance of the method in production. Of the 29,500 samples tested, 80 produced a positive IA result. Analysis by GC-MS confirmed the presence of at least 1 BZD compound in 61 of the samples corresponding to a confirmation rate of 76%. The Online BZD IA modified by the automatic addition of beta-glucuronidase appears well adapted for the rapid detection of BZDs and their metabolites in human urine.

Simultaneous HPLC Separation of Diastereoisomers of the Oxazepam Glucuronide and Oxazepam. Application to the Study of Urinary Excretion

Abstract The authors present a simple liquid chromatographic method which enables direct separation of S(+) and R(−) oxazepam glucuronides and free oxazepam as well as diazepam (internal standard) in a single run. Metabolites and oxazepam are quantified by direct injection of diluted urine. Application to the study of urinary excretion in sheep revealed a S/R ratio of 6.37 which is one of the highest ever observed among various animal species. The percentage eliminated as refered to administrated oxazepam was found to be near 35% which is particularly low compared to other species (60 to 80%).

(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.

Factors and conditions affecting the glucuronidation of oxazepam.

The aim of the present work was to investigate the impact of disease states and environmental and host factors on the glucuronidation of oxazepam. Glucuronidation represents quantitatively one of the most important metabolic conjugation pathways (phase II) in man for the inactivation and detoxication of xenobiotics and endogenous compounds and the liver is the major site for it to take place. Far less attention has been paid to the conjugation reactions in previous clinical research in this field compared to the immense interest in the oxidative biotransformation pathways (phase I). This fact is mainly due to the latter giving rise to active or reactive metabolites with a toxicological potential. The metabolism of oxazepam expresses exclusively the capacity for glucuronide formation. It was a prerequisite to establish the bioavailability of oxazepam prior to succeeding studies on the oral disposition of the drug. A preparation for intravenous administration was created. Clearance was chosen as measurement of the capacity to glucuronidate oxazepam. Severe decompensated liver disease was associated with a significant decrease in oxazepam clearance, that became even more obvious when corrected for by a diminished binding to plasma proteins. This increase in free fraction of oxazepam was substantial and could mainly be accounted for by low plasma albumin values. The results are in part a settlement with earlier studies on glucuronidation in liver disease and they may undoubtedly be ascribed to the severe degree of liver disease. For the first time it was shown that hypothyroidism led to a decline in the clearance and metabolism of oxazepam and paracetamol that is mainly biotransformed by glucuronidation. It was concluded that the enzymes responsible for glucuronidation in hypothyroidism are under the influence of thyroid hormones as is the case with oxidative enzymes. Further studies focused on the effect of host and environmental factors on glucuronidation. A commercially available very low calorie product for the treatment of obesity resulted in a decrease in oxazepam clearance and a lack of co-factors as a consequence of the low calorie intake was explanatorily proposed. Beta-adrenoceptor antagonists are often prescribed together with other drugs and close knowledge on interactions is mandatory but insufficient in regard of drugs being glucuronidated. Despite the mutual metabolic pathway labetalol exerted no dispositional alterations concerning oxazepam. It was moreover suggested that very elderly subjects between the age of 80 to 94 years had a reduced clearance of oxazepam.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of Oxazepam Glucuronide in Human Urine by Liquid Chromatography/APCI-Mass Spectrometry.

A high performance liquid chromatography-mass spectrometric (LC-MS) method for the determination of oxazepam glucuronide in the human urine was investigated. Oxazepam glucuronide was extracted from the urine by using Sep-Pak C18 cartridge with methanol-50 mM ammonium acetate (70 : 30 v/v) as eluent. The eluate was analyzed by LC-MS equipped with atmospheric pressure chemical ionization (APCI). High performance liquid chromatographic (HPLC) analyses were performed on a L-column (C18 reversed phase column) in the solvent system of methanol-50 mM ammonium acetate (70 : 30 v/v) at a flow rate of 1.0 ml/min. The vaporizer temperature was set to 230°C, desolvation temperature to 390°C, drift voltage to 108V, focus voltage to 96V and multiplier voltage to 2400V. Peak areas were measured using selected ion monitoring (SIM) of a base ion peak at m/z 287. The calibration curve gave a good linearity over the range of 0.25-1 μl/ml. The detection limit was 50 ng/ml by recording the base ion peak at m/z 287.

Effects of protein and carbohydrate content of diet on drug conjugation

Eight healthy subjects were fed a high-protein-low-carbohydrate diet and, after a 3-day washout period, an isocaloric low-protein-high-carbohydrate diet. They received acetaminophen and oxazepam, drugs metabolized primarily by conjugation, on days 11 and 13, respectively, of each diet. Changing the diets of subjects from the high-protein-low-carbohydrate diet to the low-protein-high-carbohydrate diet resulted in a 14% increase in urinary recovery of acetaminophen glucuronide and a 32% increase in urinary recovery of oxazepam glucuronide (p less than 0.05). The increases in glucuronidation were at the expense of other pathways of metabolism, and there were no significant changes in the metabolic clearance rates of acetaminophen and oxazepam. Mean renal clearances of acetaminophen glucuronide, acetaminophen sulfate, and oxazepam glucuronide decreased 45%, 32%, and 54%, respectively (p less than 0.05), when the subjects were switched to the low-protein-high-carbohydrate diet.

Physiological modeling of drug and metabolite: Disposition of oxazepam and oxazepam glucuronides in the recirculating perfused mouse liver preparation

The disposition of tracer doses of 3H-oxazepam was studied in the recirculating perfused mouse liver preparation. 3H-Oxazepam was biotransformed primarily to the diastereomeric 3H-oxazepam glucuronides, which either effluxed into the circulation or underwent biliary excretion. Three additional, unknown metabolites constituted a small fraction (5-10%) of the total radioactivity recovered in bile (7% of dose); no other metabolite was detected in perfusate. A physiologically based model, comprising the reservoir, liver blood and tissue, and bile, was fitted to reservoir concentrations of 3H-oxazepam and 3H-oxazepam glucuronides, and the cumulative amount excreted into bile. The model allowed for consideration of elimination pathways other than glucuronidation and the presence of a transport barrier for the oxazepam glucuronides across the hepatocyte membrane. The fitted results suggest a slight barrier existing for the transport of metabolites across the sinusoidal membrane, inasmuch as the transmembrane clearance was comparable to liver blood flow rate. Upon further comparison of estimates of formation, biliary, and transmembrane clearances for the oxazepam glucuronides, the rate-limiting step in the overall (biliary) clearance appears to be a poor capacity for biliary excretion. The influence of the cumulative volume loss that a recirculating perfused organ system incurs upon repeated sampling was discussed, and a compartmental method of correcting the observed concentrations of drug and generated metabolite was presented.