Toxicology Department, Cuyahoga County Medical Examiner's Office (CCMEO), 11001 Cedar Avenue, Cleveland, OH 44106, USA.
Department of Chemistry, The University of Akron, 190 E. Buchtel Avenue, Akron, OH 44325, USA.
J Anal Toxicol. 2020 Oct 12;44(7):708-717. doi: 10.1093/jat/bkaa072.
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 0.5 mL 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 20 μg/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 30 min 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 met all 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.
建立并验证了一种通过固相萃取-液相色谱-串联质谱法检测血液和尿液中 40 种苯二氮䓬类药物、（±）佐匹克隆、扎来普隆和唑吡坦的分析方法。在 29 种分析物中，有 29 种在 0.5 mL 全血中进行定量，用于调查死后、药物辅助性性侵犯（DFSA）和药物影响下驾驶（DUID）案件。使用七点、线性、1/x 加权校准曲线，七个浓度点的校准曲线范围为 2、5、10 和 20μg/L，涵盖了我们在死后、DFSA 和 DUID 样本中遇到的大多数情况。除了氟甲喹唑仑的羟基代谢物α-羟基氟拉卓仑外，所有分析物都有参考物质。根据氟甲喹唑仑的裂解模式预测了α-羟基氟拉卓仑的裂解，并且为该分析物添加了适当的转换，以实现对其的监测。尿液样本在 55°C 下用经过基因修饰的β-葡萄糖醛酸酶水解 30 分钟，根据奥沙唑仑葡萄糖醛酸苷的测定，效率>95%。广泛的样品制备包括渗透裂解和蛋白质沉淀与甲醇/乙腈混合物结合，然后冷冻和离心，从而产生异常高的信噪比。质量控制（QC）的偏差和批内/批间精密度均在±15%以内，除了氯硝西泮和依替唑仑在±20%以内。所有 43 种用于 QC 性能测试的分析物均在定量报告范围内符合定量报告标准，并且在仅存在于校准液中的 14 种分析物中定性报告。25 种分析物符合所有定量报告标准，包括稀释完整性。在同一批中分析定量血液和定性尿液样本的能力是该程序最有用的元素之一。在过去的 6 个月中，我们实验室在 300 多个样本的分析中常规使用了这种灵敏、特异和稳健的分析方法。
