应用液相色谱-高分辨离子轨道阱质谱法检测人干血点中的睾酮酯

The Analysis of Testosterone Esters in the Dried Blood Spot by Liquid Chromatographic Q Exactive High Resolution Mass Spectrometry

目的:建立从干血点中提取并检测睾酮酯类兴奋剂的方法,以解决辨别外源性睾酮的难题。方法:取20μL的干血点样品,经过水复溶后,对蛋白进行沉淀,利用有机溶剂进行液液萃取,使用甲氧胺试剂进行衍生化,用高分辨液相色谱质谱进行检测分析。结果:本方法对醋酸睾酮酯、丙酸睾酮酯、苯甲酸睾酮酯、苯丙酸睾酮酯、环戊丙酸睾酮酯、庚酸睾酮酯、癸酸睾酮酯、十一酸睾酮酯的检测限为0.1~0.3 ng/mL,提取回收率为50.0%~86.0%。采用液相色谱实现了8种睾酮酯的肟化产物的同分异构体的基线分离,并对E和Z型异构体进行了结构推测,满足世界反兴奋剂机构(WorldAnti-DopingAgency,WADA)的相关要求。人体试验中,对6名健康成年男性志愿者一次性肌肉注射0.25 g十一酸睾酮后,采用本方法可以检测到停药后7~14天的睾酮酯。结论:新方法具有快速、灵敏、微创的特点,是一种直接检测睾酮酯的方法,已通过ISO-17025质量体系认证,即将作为睾酮类兴奋剂药物的有效检测手段,在北京2022年冬奥会、冬残奥会的反兴奋剂检测中使用。

Objective:To establish an unequivocally method to analyze testosterone esters from dried blood spot and thus to discriminate exogenous with endogenous testosterone.Method:20 microliter dried blood sample was redissolved in water and then the protein was precipitated.A liquid-liquid extraction was performed and the organic phase was collected.The residue was reacted with methoxylamine,and then the Q Exactive high resolution mass spectrometer was used to analyze the sample after reconstitution.Results:The detection limit was 0.1 to 0.3 ng/mL for testosterone acetate,testosterone propionate,testosterone benzoate,testosterone phenylpropionate,testosterone cypionate,testosterone enanthate,testosterone decanoate and testosterone undecanoate.The extraction recovery rate was 50.0%to 86.0%.The oxime products of testosterone esters were baseline separated by liquid chromatographic conditions,and the E and Z isomers of oxime were tentatively identified which meet the requirements of(World Anti-Doping Agency,WADA).In human trail,six male adults were intramuscularly injected 0.25 gram testosterone undecanoate,and the detection window was 7 to 14 days.Conclusion:the new method is fast,high sensitivity and minimally invasive.It is a direct method to detect testosterone preparations,which has passed the qualification of ISO-17025 accreditation,and it will be implemented in 2022 Beijing Winter Olympic Games and Winter Paralympic Games.