Department of Chemistry and Forensic Science, University of Technology Sydney, Broadway, NSW, Australia.
J Anal Toxicol. 2010 Jun;34(5):243-51. doi: 10.1093/jat/34.5.243.
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.
β-葡萄糖醛酸酶常用于苯二氮䓬类药物尿液药物检测中使β-葡萄糖醛酸苷脱共轭。人们普遍认为，与酸催化水解相比，使用β-葡萄糖醛酸酶水解是首选方法，因为酸催化水解已知会诱导苯二氮䓬类药物的降解和转化。然而，迄今为止，文献尚未报道任何由商业β-葡萄糖醛酸酶产品引发的苯二氮䓬类药物转化的案例。在这项研究中，将含有奥沙西泮或奥沙西泮葡萄糖醛酸的尿样与来自大肠杆菌、海蜗牛和普通滨螺的β-葡萄糖醛酸酶在各种孵育条件下孵育。经过液液萃取后，通过液相色谱-串联质谱法和气相色谱-质谱法分析提取物中是否存在苯二氮䓬类药物。检查的三种酶制剂都能够将奥沙西泮或奥沙西泮葡萄糖醛酸转化为去甲西泮（去甲安定）。去甲西泮的形成与孵育温度、孵育时间、奥沙西泮浓度和酶浓度呈正相关。在所有研究的酶水解条件下，去甲西泮的形成百分比相对于系统中存在的奥沙西泮的量<2.5%。本研究的结果具有临床和法医学意义，很明显，对于涉及酶催化水解的测试中生物样本中去甲西泮的检测，应谨慎解释。
