According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were −10.0% and 1.4% for fentanyl, −2.1% and −0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from −8.5% to −19.9% for the low concentrations, −3.6% to −14.7% for the medium concentrations and 1.5% to −16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5–100 and 4-ANPP had a linear range of 0.4–80 μg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3 μg/kg with a mean of 149.2 μg/kg (n = 10). The range of norfentanyl concentrations were 1.9 to 50.0 μg/kg with a mean of 14.1 μg/kg (n = 10). The range of 4-ANPP concentrations were 3.2 to 23.7 μg/kg with a mean of 7.5 μg/kg (n = 7).

中文翻译：

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使用经过验证的QuEChERS提取和LC-MS-MS分析定量检测肝组织中的芬太尼和代谢产物。

根据国家药物滥用研究所（NIDA）的数据，每天有一百多人死于阿片类药物过量。随着有效的合成阿片类药物（如芬太尼）的可用性增加，过量死亡人数增加了。法医验尸毒理标本通常处于分解的各个阶段，经历自溶和腐烂，这使提取变得复杂，对毒理学家造成了困难的挑战。在创建有效且简化的分析方案的同时，隔离目标药物是大多数毒理学实验室的目标。本研究提出了一种快速，简便，廉价，有效，坚固而安全的提取方案的验证，作为有效提取和检测芬太尼及其主要代谢物的替代分析方法：去甲芬太尼和去丙酰芬太尼（4-ANPP）。按照美国法医学会标准委员会（ASB）标准036提出的要求，对采用串联质谱分析的液相色谱法进行了验证。评估的参数包括选择性，基质效应（MEs），线性，处理过的样品稳定性，偏差，精度和使用来自真实死后病例的肝样品的适用性证明。在低浓度和高浓度下，芬太尼的ME（表示为电离抑制或增强百分比）分别为-10.0％和1.4％，4-ANPP分别为-2.1％和-0.3％和去甲芬太尼分别为3.1％和2.8％。三种分析物的偏差在低浓度范围为-8.5％至-19.9％，在中等浓度范围为-3.6％至-14.7％，在高浓度范围为1.5％至-16.1％，所有范围均在±20％的指导范围内。三种分析物的精密度范围为2.2％至15.1％。芬太尼和去甲芬太尼的线性范围为0.5–100，而4-ANPP的线性范围为0.4–80μg/ kg。真实的死后肝脏样品的芬太尼浓度范围为56.6至462.3μg/ kg，平均为149.2μg/ kg（n  = 10）。去甲芬太尼浓度范围为1.9至50.0μg/ kg，平均值为14.1μg/ kg（n  = 10）。4-ANPP浓度范围为3.2至23.7μg/ kg，平均值为7.5μg/ kg（n  = 7）。