超高效液相色谱串联质谱同时检测人血清5种甾体激素方法学及生物参考区间的建立

目的 建立超高效液相色谱串联质谱(UPLC-MS/MS)同时测定人血清中睾酮(T)、双氢睾酮(DHT)、雄烯二酮(AD)、脱氢表雄酮(DHEA)和17α羟孕酮(17OHP)的方法，并确定表观健康人群血清中5种甾体激素的参考区间。方法 血清样品沉淀后经SOLAμHRP固相萃取柱纯化，采用Poroshell 120 ECC18色谱柱分离，用含0.05%甲酸的30%乙腈溶液和90%乙腈溶液梯度洗脱。采用电喷雾正离子多反应监测模式检测，采用同位素内标法定量检测甾体激素的浓度。对建立的UPLC-MS/MS方法进行全面的性能验证，并建立适用于上海地区人群人血清5种甾体激素的参考区间。结果 UPLC-MS/MS检测人血清中5种甾体激素的最低定量限为0.01～0.10 ng/mL，标准曲线范围覆盖1 000倍，r²值均>0.999 5，线性良好，样本经稀释后可报告范围覆盖5 000倍。5种分析物批内与批间的准确度为95.0%～105.6%，精密度均<8%。8种结构类似物对目标分析物均无干扰，高三酰甘油不影响检测结果，溶血可导致DHEA检测值降低。各分析物的提取回收率为...

Simultaneous determination of 5 steroid hormones in human serum by ultra-performance liquid chromatography-tandem mass spectrometry and establishment of reference intervals in healthy adults

Objective To establish and validate a method for the simultaneous determination of testosterone(T),dihydrotestosterone(DHT),androstenedione(AD),dehydroepiandrosterone(DHEA)and 17alphahydroxyprogesterone(17OHP)in human serum by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),and to establish the reference intervals of 5 steroid hormones in healthy adults.Methods The serum samples were purified by SOLAμHRP solid-phase extraction,and they were separated by Poroshell 120 EC-C18 analytical column.Mobile phase was constituted of 30%acetonitrile solution and 90%acetonitrile solution containing 0.05%formic acid with gradient elution.The electrospray positive ion multi-reaction monitoring mode was used for determination,and the isotope internal standard method was used to quantify the concentration of steroid hormones.The performance of the method was verified comprehensively,and the reference intervals suitable for the local population in Shanghai were established.Results The minimum limits of quantification of the 5 steroid hormones in human serum were 0.01-0.10 ng/mL.The standard curve range covered 1000 times(r²≥0.9995)and showed good linearity,and the sample after dilution can be reported to cover the range of 5000 times.The average accuracy of 5 steroid hormones ranged from 95.0%to 105.6%,with the precision of<8%.None of the 8 structural analogs interfered with the target analytes,high triglyceride blood did not affect the determination results,and hemolysis resulted in low DHEA concentration.The extraction recoveries of the analytes were 60.5%-93.3%,and there was no obvious matrix effect after calibration of internal standard.Analytes demonstrated good stability at room temperature for 4 h,at-25--15℃for 30 d or at-90--70℃for 100 d.The method was used and participated in a survey of endocrine accuracy verification supplied by the National Center for Clinical Laboratories in 2019.The determination deviations of T and 17OHP were-5.00%-0.38%.The results of 367 physical examination subjects were analyzed and showed that there was statistical significance in the concentration of 5 steroid hormones between females and males(P<0.05).There was statistical significance between the 2 age subgroups(aged 16-50 years and aged>50 years)of female groups(P<0.05).Conclusions A UPLC-MS/MS has been established for the determination of 5 steroid hormones in human serum,and the reference intervals suitable for the Central Laboratory of Shanghai Xuhui Central Hospital have been verified.