用液相色谱-串联质谱联用技术检测4种刺激剂类禁用物质

The Developed LC-MS/MS Method for the Detection of Four Prohibited Stimulants

目的：使用液相色谱-串联质谱联用技术,为世界反兴奋剂机构（WADA）在检测窗口中拟增加的4种刺激剂甲磺美嗪（amezinium methylsulfate,I）、吗拉宗（morazone,II）、贝美格（3-ethyl-3-methylglu-tarimide,III）和阿拉明（metaraminol,IV）建立了常规检测方法,并对方法进行考察和验证。方法：用5%的醋酸铅水溶液将样品中蛋白质等大分子沉淀后,离心10分钟,取上清液上机检测。质谱采用电喷雾电离源（ESI）,在正离子模式下,通过多反应监测（MRM）模式对样品进行分析。结果：经过定性和定量分析,得到上述四种刺激剂的检测限（LOD）分别为0.1、0.5、5和0.5 ng/m L;定量限（LOQ）分别为0.3,1.5,15和1.5 ng/m L;线性范围（r2〉0.99）分别在0.3~5000、1.5~3000、15~3000和1.5~4500 ng/m L之间。每种刺激剂在低、中和高3个浓度范围的基质效应均在98%~120%之间;准确度误差在9%以内;日内精密度的标准偏差低于7.7%,日间精密度的标准偏差不超过10%。结论：本研究建立的检测方法具有样品前处理简单、灵敏度高、准确度好和基质干扰较小等特点,满足兴奋剂检测实验室的要求,并已经应用于常规检测中。

Objective To develop and validate a liquid chromatography-tandem mass spectrometry（LC-MS/MS）method for the detection of 4 stimulants including amezinium methylsulfate（I）,morazone（II）,3-ethyl-3-methylglutarimide（III）and metaraminol（IV）to be added to the detection window by World Anti-doping Agency. Method An equal volume of 5% lead acetate aqueous solution was employed to pre-cipitate the proteins in urine samples. After centrifugation for 10 min,the supernatant was introduceddirectly into the LC-MS/MS system using positive electrospray ionization with multiple reaction monitor-ing（MRM）mode. Results The limit of detections（LODs）for stimulants I-IV were 0.1,0.5,5 and 0.5 ng/m L respectively,while the limit of quantifications（LOQs）were 0.3,1.5,15 and 1.5 ng/m L accordingly.The assays were linear（r2〉0.99）over the concentration ranges of 0.3-5000,1.5-3000,15-3000 and 1.5-4500 ng/m L in human urine respectively. The established method also achieved satisfactory results interms of intra- and inter-day precisions（CV〈10%）,accuracies（bias〈9%）,matrix effect（98-120%）andspecificity at low,medium and high concentration levels. Conclusions The developed method is precise,sensitive and accurate with minimum sample preparation. It is a fit- for- purpose approach and hasbeen applied in the National Anti-Doping Laboratory for doping-control sample analysis.