目的:生物样本的毒理学分析在法医和临床研究中发挥重要作用。摄入的药物作为与内源性物质如葡萄糖醛酸的缀合物在尿液中排泄;水解这些缀合物改善了通过液相色谱-串联质谱法(LC-MS/MS)对目标药物的测定。在这项研究中,我们试图改善五种精神活性药物(11-nor-9-羧基-Δ9-四氢大麻酚,奥沙西泮,劳拉西泮,替马西泮,和阿米替林)。
方法:通过改变温度优化尿液中葡糖苷酸结合物的酶促水解效率,酶体积,和反应时间。水解直接在萃取柱上进行。这种使用LC-MS/MS的分析方法在经过彻底验证后应用于法医尸检样品。
结果:我们发现重组β-葡糖醛酸糖苷酶B-One®在室温下直接在萃取柱上在3分钟内定量水解这些缀合物。这种柱上方法节省了时间,并消除了转移到萃取柱期间有价值样品的损失。用这种方法处理的基于LC-MS/MS的校准曲线显示出良好的线性,r2值超过0.998。该方法的日内和日间准确度和准确度分别为93.0-109.7%和0.8-8.8%,分别。回收率在56.1-104.5%范围内。基质效应在78.9和126.9%之间。
结论:我们建立了直接在提取柱上酶水解葡萄糖醛酸苷结合物后尿液中五种精神活性药物的LC-MS/MS方法。该方法已成功应用于法医尸检样本。所建立的方法将具有广泛的应用,包括法医和临床毒理学调查。
OBJECTIVE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline).
METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation.
RESULTS: We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%.
CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.