Abstract：
The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 μL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 μL methanol:acetonitrile (3:1 v/v) added with 1.5 μL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 μL methanol, and 1 μL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.
摘要：
由于在中毒和过量病例中反复报道了它们，因此越来越需要在生物流体中检测硝基苯。开发并验证了一种简单的方法，用于定量干血斑（DBS）中低水平的九种硝基苯类似物和布鲁芬。将10μL的加标全血沉积在Capitainer®B卡上并使其干燥。这个地方被冲出去了，并用500μL甲醇:乙腈(3:1v/v)萃取,加入1.5μL芬太尼-D5作为内标。搅拌后,超声处理，和小瓶的离心，溶剂在氮气下干燥，提取物在30μL甲醇中重建，并将1μL注射到UHPLC-MS/MS仪器中。方法验证显示线性校准在1-50ng/mL范围内，LOD值范围在0.3ng/mL（异氮烯）和0.5ng/mL（布鲁芬）之间，所有化合物的平均CV%和偏差%在15%和10%以内，分别。由于血液和滤纸成分的基体效应在85-115%内,而回收率在15-20%之间。针对时间和温度的稳定性测试显示,对于长达28天的储存期没有显著变化。室温被证明是最好的样品储存条件。UHPLC-MS/MS被证明能够可靠地识别低浓度的所有目标分析物，即使在小样本体积，从DBS卡获得的，这反过来又被证实是有效和可持续的微量取样装置。该程序提高了毒理学测试的效率，并为鉴定硝基苯类非法化合物提供了一种创新方法。
