高效液相色谱串联质谱法同时定量测定人血清中脱氢表雄酮、睾酮及雄酮

目的: 建立一种快速、灵敏地同时测定人血清中脱氢表雄酮、睾酮及雄酮的高效液相色谱串联质谱(电喷雾电离模式)定量方法。 方法: 血清样品选用甲基叔丁基醚(MTBE)萃取后,取上清液用盐酸羟胺对3种激素进行柱前衍生化后进样测定。色谱柱为Waters YMC ODS-AQ(2.0 mm×150 mm,3.0 μm),流动相为70%乙腈和30%醋酸(0.02%)溶液,流速为0.2 mL· min^-1,柱温为50 ℃。本文采用电喷雾离子源进行正离子离子化,使用MRM检测模式选择性监测脱氢表雄酮 (m/z 304.2/253.2)、睾酮(m/z 304.2/124.0)、雄酮 (m/z 306.2/255.2)及内标炔诺酮(m/z 314.1/124.2)。 结果: 脱氢表雄酮、睾酮与雄酮的最低定量限(LLOQ)分别为0.10,0.05,0.10 ng·mL^-1;标准曲线相关系数(r)均大于0.99;3种激素的日间及日内方法回收率均在±115%之内,日内及日间精密度(RSD)均小于15%。 结论: 实现在ESI电离模式下,用HPLC-MS/MS 同时测定脱氢表雄酮、睾酮与雄酮3种甾体激素;本方法具有良好的特异性、灵敏度、准确度及精密度,能满足人血清样品定量分析需要。

Objective: To establish a rapid and sensitive method for simultaneous quantitative determination of dehydroepiandrosterone (DHEA),testosterone (T) and androsterone (ADT) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) equipped with electrospray (ESI) ionization interface. Methods: Dehydroepiandrosterone,testosterone,androsterone,and internal standard (norethisterone) were extracted from human serum using MTBE (methylterbutylethylene) followed by treatment with hydroxylamine hydrochloride to produce derivatives before determination.Analytes were separated on Waters YMC ODS-AQ(2.0 mm×150 mm,3.0 μm) with the mobile phase of acetonitrile and 0.02%acetic acid (70∶30).The flow rate was 0.2 mL·min^-1,and the column temperature was 50 ℃,resulting the well separation of chromatography peaks.The electrospray ionization method and MRM detection mode with positive ion were adopted to selectively detect DHEA (m/z 304.20/253.20),T(m/z 304.2/124.0),ADT (m/z 306.2/255.2)and internal standard norethisteron (m/z 314.1/124.2). Results: The results indicated the lower limits of quantitation of DHEA,T and ADT were 0.10,0.05 and 0.10 ng·mL^-1,respectively.All of correlation coefficients of standard curves(r) were higher than 0.99;the intra day/inter day method recoveries and precisions (RSD) in determining these three hormones were within 115% and less than 15%,respectively. Conclusion: Our method provided an efficient determination method for the detection of three kinds of hormones (DHEA,T and ADT) in human serum using HPLC-MS/MS with ESI interface.This method is specific,sensitive,accurate and precise,satisfying the quntificaiton requirements for human serum samples.