Department of Forensic and Investigative Science, West Virginia University, 1600 University Avenue, Morgantown, WV 26506, USA.
Department of Pathology, Allegheny General Hospital, 320 E. North Avenue, Pittsburgh, PA 15212, USA.
J Anal Toxicol. 2021 Jan 21;44(9):957-967. doi: 10.1093/jat/bkaa006.
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).
根据国家药物滥用研究所（NIDA）的数据，每天有超过 100 人死于阿片类药物过量。随着强效合成阿片类药物（如芬太尼）的供应增加，过量致死人数有所上升。法医死后毒理学标本通常处于不同程度的分解阶段，经历自溶和腐烂，这增加了提取的难度，给毒理学家带来了挑战。对于大多数毒理学实验室来说，分离目标药物的同时创建一个高效且简化的分析方案是一个目标。本研究提出了一种快速、简便、廉价、有效、坚固和安全的提取方案，作为一种有效的提取和检测芬太尼及其主要代谢物：去甲芬太尼和去丙酰芬太尼（4-ANPP）的替代分析方法。液质联用分析方法按照美国法医学科学院标准委员会（ASB）标准 036 提出的要求进行了验证。评估的参数包括选择性、基质效应（MEs）、线性、处理后样品稳定性、偏差、精密度和使用真实死后案例的肝样本来证明适用性。在低浓度和高浓度时，芬太尼的 ME（表示为电离抑制或增强的百分比）分别为-10.0%和 1.4%，4-ANPP 为-2.1%和-0.3%，去甲芬太尼为 3.1%和 2.8%。三种分析物的偏差在低浓度时为-8.5%至-19.9%，在中浓度时为-3.6%至-14.7%，在高浓度时为 1.5%至-16.1%，均在±20%的范围内。三种分析物的精密度在 2.2%至 15.1%之间。芬太尼和去甲芬太尼的线性范围为 0.5-100，4-ANPP 的线性范围为 0.4-80μg/kg。真实的死后肝样本文芬太尼浓度范围为 56.6-462.3μg/kg，平均浓度为 149.2μg/kg（n=10）。去甲芬太尼浓度范围为 1.9-50.0μg/kg，平均浓度为 14.1μg/kg（n=10）。4-ANPP 浓度范围为 3.2-23.7μg/kg，平均浓度为 7.5μg/kg（n=7）。
