You Youwen, Proctor Rachel M, Haughan Joanne, Missanelli Jaclyn R, Robinson Mary A
Department of Clinical Studies, University of Pennsylvania, School of Veterinary Medicine, New Bolton Center Campus, 382 W. Street Rd., Kennett Square, PA 19348, USA.
Pennsylvania Equine Toxicology & Research Laboratory, 220 East Rosedale Avenue, West Chester, PA 19382, USA.
J Anal Toxicol. 2023 Apr 14;47(4):393-402. doi: 10.1093/jat/bkad008.
Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitative and confirmatory liquid chromatography--tandem mass spectrometry (LC-MS-MS) method was developed for PMA analysis in equine urine. PMA was extracted by solid phase extraction, separated on a C18 column and detected using a triple quadrupole mass spectrometer. The mass spectrometer was operated in positive-ion mode, and multiple reaction monitoring was used to monitor product ions m/z 188, m/z 281 and m/z 323. The method was validated for extraction recovery, matrix effect, specificity, sensitivity, precision and accuracy, carryover and processed sample stability according to the guidelines of the US Food and Drug Administration for bioanalysis. The limits of detection and quantification were 5 and 10 pg/mL, respectively. Linearity was obtained over the concentration range of 10-10,000 pg/mL. To confirm PMA in equine urine, LC retention time, diagnostic product ions (m/z 188, m/z 281 and m/z 323) and product ion ratio were used as the criteria. The lowest concentration for confirmatory analysis was validated at 50 pg/mL. The method was applied to measure the PMA concentrations in equine urine following intravenous administration of fentanyl to a research horse and has confirmed the presence of PMA in post-race urine samples. This method is a valuable addition to the arsenal of equine doping control methods to combat illegal doping and protect racehorse health.
芬太尼是一种强效的合成μ阿片受体激动剂，被国际赛马委员协会和国际马术联合会禁止用于马术运动。在作者所在实验室对赛马赛后常规兴奋剂筛查中，已证实马血中存在芬太尼。虽然芬太尼可在血液中被检测和确认，但其代谢迅速，因此预计对马尿中的代谢物N-[1-(2-苯乙基-4-哌啶基)]马来酰苯胺酸（PMA）进行筛查可延长检测时间。在本研究中，开发了一种用于马尿中PMA分析的定量和确证液相色谱-串联质谱（LC-MS-MS）方法。PMA通过固相萃取进行提取，在C18柱上分离，并使用三重四极杆质谱仪进行检测。质谱仪以正离子模式运行，采用多反应监测来监测m/z 188、m/z 281和m/z 323的产物离子。根据美国食品药品监督管理局生物分析指南，对该方法的提取回收率、基质效应、特异性、灵敏度、精密度和准确度、残留以及处理后样品稳定性进行了验证。检测限和定量限分别为5和10 pg/mL。在10 - 10,000 pg/mL的浓度范围内获得了线性关系。为确证马尿中的PMA，将LC保留时间、诊断产物离子（m/z 188、m/z 281和m/z ！323）以及产物离子比率用作标准。确证分析的最低浓度在50 pg/mL时得到验证。该方法应用于测定向一匹实验用马静脉注射芬太尼后马尿中的PMA浓度，并已证实在赛后尿样中存在PMA。该方法是打击非法使用兴奋剂和保护赛马健康的马用兴奋剂控制方法中的一项宝贵补充。
