Department of Public Health, Experimental and Forensic Medicine, University of Pavia, via Forlanini, 12, 27100, Pavia, Italy.
Curr Pharm Des. 2022;28(32):2622-2638. doi: 10.2174/1381612828666220831103224.
The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis.
A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases.
100 μL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode.
25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine.
The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.
在生物体液和组织中检测新型苯二氮䓬类药物（包括传统苯二氮䓬类药物），可以为进行临床和法医毒物分析的实验室提供重要的分析更新。
本研究建立并充分验证了一种液相色谱-串联质谱法（LC-MS/MS），并将其应用于一组接受戒断治疗的患者和中毒病例的真实尿液样本。
将 100μL 尿液加入含氘代内标物的缓冲液中；然后通过液液萃取程序提取样品，在氮气流下干燥，并在流动相中重新溶解。采用 C18 反相柱进行梯度洗脱，实现色谱分离。质谱采用正离子极化模式和多反应监测模式进行检测。
对 25 种化合物进行了仪器分析优化：9 种新型苯二氮䓬类药物和 16 种传统化合物（母体药物及其主要代谢物）。对所有化合物进行了灵敏度、特异性、线性、准确度、精密度、回收率、基质效应和交叉污染的评估。只有西拉西泮未满足所有验证标准。在分析的 50 个样本中，有 10 个样本至少有一种监测到的化合物呈阳性。特别是，从同一病例采集的两个不同样本中，均检测到了新型苯二氮䓬类药物氟硝西泮呈阳性。
该方法不仅可用于检测传统苯二氮䓬类药物，还可用于检测新型苯二氮䓬类药物。在真实样本中鉴定出一种新的精神活性物质，证实了分析程序应进行更新，以尽可能多地纳入更多物质。
