Enzymatic digestion, solid-phase extraction, and gas chromatography/mass spectrometry of derivatized intact oxazepam in urine

Langner, J G; Gan, B K; Liu, R H; Baugh, L D; Chand, P; Weng, J L; Edwards, C; Walia, A S

doi:10.1093/clinchem/37.9.1595

Cited by 22 publications

(4 citation statements)

References 13 publications

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“…For example, the drug substance in Serepax® is oxazepam which is a metabolite of diazepam (marketed as Valium®). Detection of oxazepam in a biological fluid may not necessarily be an indication of oxazepam use, as it could be a metabolite of temazepam, diazepam and many other drugs such as clorazepate, prazepam, and medazepam (8). Diazepam, on the other hand, can be a thermal degradation product of ketazolam (9), as well as a metabolite of tetrazepam (10).…”

Section: Introductionmentioning

confidence: 99%

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

Lewis

Wang

et al. 2010

Journal of Analytical Toxicology

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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.

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Section: Introductionmentioning

confidence: 99%

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

Lewis

Wang

et al. 2010

Journal of Analytical Toxicology

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“…Examples of other agents that produce these effects include oxazepam, loratadine, and doxylamine succinate. All of these agents have been shown to induce CYP2B1/2 and are considered PB-like inducers based on changes in hepatic cytochrome P450 enzyme levels (Langer et al, 1991;Griffin et al, 1996;Parkinson et al, 1992;Bookstaff et al, 1995;Skare et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Octamethylcyclotetrasiloxane (D4) as an Inducer of Rat Hepatic Microsomal Cytochrome P450, UDP-Glucuronosyltransferase, and Epoxide Hydrolase: A 28-Day Inhalation Study

McKim

1998

Toxicological Sciences

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Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

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“…Consequently, detection of temazepam is often accompanied by the presence of nordiazepam and oxazepam from diazepam ingestion. Temazepam could also be a metabolite of other drugs such as clorazepate, prazepam, and medazepam [8]. Diazepam, on the other hand, could be a metabolite of tetrazepam [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis

Molnár

Bowron³

et al. 2011

Anal Bioanal Chem

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It has been previously reported that treatment of urinary oxazepam by commercial β-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid-liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant--approximately 1% (0.59-1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.

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Enzymatic digestion, solid-phase extraction, and gas chromatography/mass spectrometry of derivatized intact oxazepam in urine

Cited by 22 publications

References 13 publications

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

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

Evaluation of Octamethylcyclotetrasiloxane (D4) as an Inducer of Rat Hepatic Microsomal Cytochrome P450, UDP-Glucuronosyltransferase, and Epoxide Hydrolase: A 28-Day Inhalation Study

Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis

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