摘要
目的:兽用麻醉剂替来他明为新型毒品替代物,在全球多地区出现滥用情况,对公众健康造成巨大危害,但其检测方法的灵敏度难以满足法医学实践需求。本研究旨在建立一种测定人体生物样本中替来他明及其代谢物去乙基替来他明的超高效液相色谱-串联质谱(ultra-high performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)法,并通过方法学验证其能否应用于法医学实践。方法:选择SKF525A为内标,采用乙腈(含1 ng/mL SKF525A)萃取生物样本,漩涡振荡10 min,超声处理20 min,以10000 r/min离心10 min,取500μL上清液经0.22μm滤膜过滤,采用高效液相色谱系统(ACQUITY UPLC H-Class PLUS)和三重四极杆质谱仪(XEVO TQ-S Micro)进行检测。色谱条件采用ACQUITY UPLC?BEH C18(1.7μm,2.1 mm×100 mm)色谱柱,0.3 mL/min流速,考察4种流动相体系(20 mmol/L乙酸铵和0.1%甲酸水溶液+甲醇、20 mmol/L乙酸铵和0.1%甲酸水溶液+乙腈、水+甲醇、水+乙腈)对替来他明及去乙基替来他明的分离效果。质谱条件采用电喷雾离子源正离子模式(electrospray ionization in positive mode,ESI+)、多反应监测模式(multiple reaction monitoring,MRM),替来他明和去乙基替来他明分别选用质荷比为224.043→179.016、196.08→151.06作为定量离子对。以添加0.1、0.2、0.5、1、2、5、10、20、50、100、200 ng/mL替来他明的血样的质谱响应值为纵坐标,替来他明浓度为横坐标,通过最小二乘法进行线性回归获得校准曲线,考察替来他明在血样中的线性范围、检出限和定量限;选取替来他明低(5 ng/mL)、中(20 ng/mL)、高(100 ng/mL)3种浓度制备多份血液、肝脏及肾脏的阳性添加样本和空白样本,经前处理后上机进行检测分析并记录其响应值,考察替来他明在不同生物样本中的精密度、准确度、基质效应、提取回收率和稳定性。最后,采用UPLC-MS/MS定量检测实际案例获取的替来他明样本,以验证其适用性。结果:建立的UPLC-MS/MS法可同时检测人体生物样本中的替来他明及代谢物去乙基替来他明,保留时间分别为3.42 min和2.82 min。以流动相A(20 mmol/L乙酸铵和0.1%甲酸水溶液)和流动相B(乙腈)梯度洗脱替来他明和去乙基替来他明的效果最佳。血液样品中替来他明在0.1~200 ng/mL范围内线性关系良好,相关系数r=0.992,决定系数R2=0.983,检出限为0.03 ng/mL,定量限为0.1 ng/mL,提取回收率为92%~107%,基质效应为71%~99%;肝脏样本中的提取回收率为91%~98%,基质效应为69%~96%;肾脏样本中的提取回收率为93%~104%,基质效应为72%~100%。本方法在不同样本中的日内、日间精密度[以相对标准偏差(relative standard deviation,RSD)表示]和准确度[以相对误差(relative error,RE)表示]均控制在15%以内,且样本在-20℃冻存条件下表现出良好的稳定性。从实际案件获取的生物样本中检测到替来他明成分:案例1血液、肝脏、肾脏样本含量分别为0.37μg/mL、0.15μg/g、0.11μg/g,案例2血液样本含量为8.75 ng/mL;在血样中检测到代谢物去乙基替来他明成分。结论:UPLC-MS/MS法高效、准确、灵敏,适用于人体生物样本中替来他明和代谢物去乙基替来他明成分的检测。
Objective:Tiletamine,a veterinary anesthetic,has emerged as a novel psychoactive substance and has been abused in many parts of the world,causing great harm to public health.However,the sensitivity of existing detection methods cannot meet the needs of forensic practice.This study aims to establish an ultra-high-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the determination of tiletamine and its metabolite desethyltiletamine in human biological samples,and to verify its applicability in forensic practice.Methods:SKF525A was used as the internal standard.Biological samples were extracted with acetonitrile containing 1 ng/mL SKF525A,vortexed for 10 min,ultrasonicated for 20 min,centrifuged at 10000 r/min for 10 min,and 500μL of the supernatant was filtered through a 0.22μm membrane.Analyses were performed using an ACQUITY UPLC H Class PLUS system and an XEVO TQ-S Micro triple quadrupole mass spectrometer.An ACQUITY UPLC®BEH C18(1.7μm,2.1 mm×100 mm)column at a flow rate of 0.3 mL/min was used,and four mobile phase systems were tested to optimize separation.Detection used positive electrospray ionization(ESI+)in multiple reaction monitoring(MRM)mode,with quantifier ion transitions of mass to charge 224.043→179.016 for tiletamine and mass to charge 196.08→151.06 for desethyltiletamine.Calibration curves were established over 0.1−200 ng/mL in spiked blood samples.The linear range,limit of detection(LOD),and limit of quantification(LOQ)were determined.Low(5 ng/mL),medium(20 ng/mL),and high(100 ng/mL)concentrations of tiletamine were spiked into blood,liver,and kidney to evaluate precision,accuracy,matrix effect,recovery,and stability.Finally,actual forensic case samples were tested to validate applicability.Results:The established UPLC-MS/MS method achieved simultaneous detection of tiletamine and desethyltiletamine in human biological samples,with retention times of 3.42 min and 2.82 min,respectively.Using mobile phase A(20 mmol/L ammonium acetate and 0.1%formic acid in water)and mobile phase B(acetonitrile)produced the best separation.In blood,tiletamine showed good linearity from 0.1−200 ng/mL(r=0.992,R2=0.983),LOD 0.03 ng/mL,LOQ 0.1 ng/mL,recovery 92%−107%,and matrix effect 71%−99%.In liver and kidney,recoveries were 91%−98%and 93%−104%,and matrix effects were 69%−96%and 72%−100%,respectively.Intra-and inter-day precision[expressed as relative standard deviation(RSD)]and accuracy[expressed as relative error(RE)]were within 15%,and samples were stable at−20℃.Tiletamine was detected in actual case samples at 0.37μg/mL(blood),0.15μg/g(liver),0.11μg/g(kidney)in case 1,and 8.75 ng/mL(blood)in case 2;desethyltiletamine was also detected in blood.Conclusion:The UPLC-MS/MS method is efficient,accurate,and sensitive,and is suitable for detecting tiletamine and desethyltiletamine in human biological samples.
作者
蔡子豪
严文广
李嘉豪
丁艳君
凌江
CAI Zihao;YAN Wenguang;LI Jiahao;DING Yanjun;LING Jiang(Department of Forensic Science,Xiangya School of Basic Medical Sciences,Central South University,Changsha 410013;Department of Rehabilitation Medicine,Third Xiangya Hospital,Central South University,Changsha 410013,China)
出处
《中南大学学报(医学版)》
北大核心
2025年第6期1002-1012,共11页
Journal of Central South University (Medical Science)
基金
国家自然科学基金(82402201)
长沙市自然科学基金(kq2402237)。
