基于双衍生-串联质谱技术检测类固醇激素研究

Study on steroids detection based on dual derivatization couple with tandem mass spectrometry

目的:建立一种双衍生法-结合串联质谱技术同时检测人血清中18种类固醇激素的方法。方法:血清样本采用羟胺和1,2-二甲基咪唑-5-磺酰氯对进行衍生处理,采用液相色谱-串联质谱法正离子选择离子监测模式(SRM)下检测,色谱柱为Kinetex■C_(8)(100 mm×2.1 mm,2.6μm),以0.1%甲酸水溶液为流动相A,0.1%甲酸甲醇溶液为流动相B,梯度洗脱(0~0.5 min,35%B;0.5~5 min,35%B→100%B;5.0~5.1 min,100%B→35%B;5.1~7 min,35%B),柱温40℃,流速0.4 mL·min^(-1),进样量20μL,采集时间为6 min。结果:18种类固醇激素的线性相关系数均>0.99,回收率均在85%~115%,精密度RSD均<15%。用该方法成功地检测了156份健康人群受试者血清样本(男性75例,女性61例)并制定了参考区间。结论:该方法可用非固相萃取法同时检测孕烯醇酮、17-羟孕烯醇酮、孕酮、17-羟孕酮、皮质酮、皮质醇、11-脱氧皮质醇、21-脱氧皮质醇、醛固酮、睾酮、雄烯二酮、脱氢表雄酮、硫酸脱氢表雄酮、皮质素、18-羟皮质酮、雌酮、雌二醇、雌三醇等18种类固醇激素。

Objective:To establish a dual derivatization method combined with tandem mass spectrometry for the simultaneous determination of 18 different steroid hormones in human serum.Methods:Serum samples were treated with hydroxylamine and 1,2-dimethylimidazole-5-sulfonyl chloride for derivatization,and the resulting compounds were analyzed by liquid chromatography-tandem mass spectrometry in positive ion selected reaction monitoring(SRM)mode.The Kinetex■C_(8)column(100 mm×2.1 mm,2.6μm)was used for the separation.Mobile phase A(0.1%acetic acid in water)and mobile phase B(0.1%acetic acid in methanol)with gradient elution(0-0.5 min,35%B;0.5-5 min,35%B→100%B;5.0-5.1 min,100%B→35%B;5.1-7 min,35%B)at the flow rate of 0.4 mL·min^(-1)were applied.Column temperature was set at 40℃.Injection volume was 20μL and collection time was 6 min.Results:The linearity correlation coefficients for all 18 steroid hormones were greater than 0.99,the recovery rates ranged from 85%to 115%,and the precision RSD was less than 15%.This method was successfully applied to the analysis of serum samples from 156 healthy subjects(75 males and 61 females),and reference intervals were established.Conclusion:This method can be used to simultaneously determine 18 types of steroid hormones,such as pregnenolone,17-hydroxypregnenolone,progesterone,17-hydroxyprogesterone,corticosterone,cortisol,11-deoxycorticosterone,21-deoxycorticosterone,aldosterone,testosterone,androstanedione,dehydroepiandrosterone,sulfated dehydroepiandrosterone,adrenocorticotropin,18-hydroxicorticotropin,estrone,estradiol,and estriol using non-solid-phase extraction.