液相色谱串联质谱法测定人体血清中的类固醇激素

Determination of Steroid Hormones in Human Serum by Liquid Chromatography Tandem Mass Spectrometry

目的:建立一种能同时测定血清中6种类固醇激素[包括孕酮(P)、17-羟基孕酮(17-OHP)、皮质醇(COL)、去氢表雄酮(DHEA)、双氢睾酮(DHT)、硫酸去氢表雄酮(DHEAS)]的快速、灵敏的高效液相色谱串联质谱(HPLC-MS/MS)方法。方法:血清样本经甲基叔丁基醚液液萃取,高速离心后上清液氮气吹干,加入复溶液后上机检测。采用安捷伦C_(18)(100 mm×2.1 mm,1.8μm)色谱柱进行分离,水相A为纯水,有机相B为纯甲醇,质谱采用电喷雾电离(ESI)模式进行离子化,多反应监测模式(MRM)扫描,同位素稀释法进行定量分析。结果:血清中6个类固醇激素检测限(LOD,以信噪比(S/N)≥3为标准)为0.03~1.25 ng·mL^(-1);定量限(LOQ,以信噪比(S/N)≥10为标准)为0.05~2.50 ng·mL^(-1);在0.05~4000 ng·mL^(-1)范围内线性关系良好,相关系数(r)≥0.9950;低浓度样本日内精密度为2.4%~6.2%;中浓度样本日内精密度为2.3%~5.7%;高浓度样本日内精密度为1.3%~5.4%。低浓度样本日间精密度为0.4%~2.6%;中浓度样本日间精密度为0.3%~3.9%;高浓度样本日间精密度为0.2%~4.7%。加标回收率为86%~106%。结论:建立一种能同时测定血清中6个类固醇激素的HPLC-MS/MS方法。该方法简单、快速、灵敏度高、特异性强,可在6.5 min之内完成分离和检测,适用于人体血清中多种类固醇激素含量的定量分析。

Objective:To establish a rapid and sensitive HPLC tandem mass spectrometry(HPLC-MS/MS)method for simultaneous determination of six steroid hormones in serum,including progesterone(P),17-hydroxyprogesterone(17-OHP),cortisol(COL),dehydroepiandrosterone(DHEA),dihydrotestosterone(DHT)and dehydroepiandrosterone sulfate(DHEAS).Methods:The Serum samples were extracted with methyl tert-butyl ether,centrifuged at high speed,dried with liquid nitrogen,redissolved and tested.An Agilent C_(18)(100 mm×2.1 mm,1.8μm)column was used for separation.Water(A)and methanol(B)were applied for gradient elution.An Electrospray ionization(ESI)mode was used for ionization,multiple reaction monitoring mode(MRM)scanning was used for quantitative analysis by an isotope internal standard method.Results:The HPLC-MS/MS method separated and detected the 6 steroid hormones in serum within 6.5 min.The limits of detection(LOD)of the 6 steroid hormones in serum were 0.03-1.25 ng·mL^(-1),based on S/N≥3.The limits of quantitation(LOQ,based on signal-to-noise ratio(S/N)≥10)ranged from 0.05-2.50 ng·mL^(-1).The linearity was good in the range of 0.05-4000 ng·mL^(-1),and the correlation coefficient(r)was≥0.9950.The intra-day precision of low,medium and high concentration samples were 2.4%-6.2%,2.3%-5.7%and 1.3%-5.4%,and the inter-day precision of low,medium and high concentration samples were 0.4%-2.6%,0.3%-3.9% and 0.2%-4.7%,respectively.The spiked recoveries were 86%-109%(acceptable standard was 80%-120%).Conclusion:The established HPLC-MS/MS method is simple,rapid,sensitive and specific,and is suitable for the quantitative analysis of various steroid hormones in human serum.