Department of Chemistry, University of Turin, Turin, Italy.
Department of Chemistry, University of Turin, Turin, Italy.
J Pharm Biomed Anal. 2024 Apr 15;241:115975. doi: 10.1016/j.jpba.2024.115975. Epub 2024 Jan 12.
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.
由于氮杂卓类物质在中毒和过量用药案例中反复被报道，因此越来越需要在生物体液中检测它们。本文建立并验证了一种简单的方法，用于在干血斑（DBS）中定量检测 9 种氮杂卓类似物和布若啡。将 10μL 加标全血滴加在 Capitainer®B 卡上，使其干燥。用打孔器从卡上取下血斑，用 500μL 甲醇：乙腈（3:1，v/v）提取，加入 1.5μL 芬太尼-D5 作为内标。在涡旋、超声和离心后，氮气流下吹干溶剂，用 30μL 甲醇重溶，取 1μL 注入 UHPLC-MS/MS 仪。方法验证结果显示，该方法在 1-50ng/mL 范围内具有线性校准，LOD 值在 0.3ng/mL（异他佐辛）和 0.5ng/mL（布若啡）之间，所有化合物的平均 CV%和偏差%均在 15%和 10%以内。由于血液和滤纸成分引起的基质效应在 85-115%之间，回收率在 15-20%之间。稳定性测试结果表明，在长达 28 天的储存期内，时间和温度对储存条件没有显著影响。室温被证明是最佳的样本储存条件。UHPLC-MS/MS 能够可靠地在低浓度下识别所有目标分析物，即使是从小样本量的 DBS 卡中获得的分析物，这反过来也证实了 DBS 卡是一种有效且可持续的微采样装置。该程序提高了毒理学检测的效率，并为识别非法氮杂卓类化合物提供了一种创新方法。
