UPLC-MS/MS法同时检测人血清中5种微量内源性激素

Determination of Five Trace Endogenous Hormones in Human Serum by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry

目的:建立一种快速、高效、同时检测人血清中睾酮(T)、雄烯二酮(AD)、脱氢表雄酮(DHEA)、孕酮(P)、17α-羟孕酮(17α-OHP)浓度的UPLC-MS/MS方法。方法:血清样品采用盐酸羟胺衍生化、以提高离子化效率,以同位素氘标记的P-d9作为内标,采用Agilent ZORBAX Eclipse Plus Phenyl-Hexyl色谱柱(2.1 mm×100 mm,3.5μm),0.1%甲酸水(A)-乙腈(B)为流动相,以0.3 mL·min^(-1)流速梯度洗脱分离后,在电喷雾离子源正离子模式下扫描,分析时间5 min。结果:在0.05~100 ng·mL^(-1)浓度范围内,5种激素呈现良好线性关系,定量下限均达到0.05 ng·mL^(-1),批内、批间精密度和准确度均符合要求,平均提取回收率和归一化基质因子变异均在15%以内。结论:该方法成功用于实际血清样本中5种激素的定量分析。

Objective:To establish a rapid and efficient ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the simultaneous determination of trace testosterone(T),androstenedione(AD),dehydroepiandrosterone(DHEA),progesterone(P)and 17α-hydroxyprogesterone(17α-OHP)in human serum.Methods:Human serum samples were derived by hydroxylamine hydrochloride to improve ionization efficiency.The isotopically labeled P-d9 was selected as internal standard.The chromatographic separation was performed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column(2.1 mm×100 mm,3.5μm)using acetonitrile and water containing 0.1%formic acid as the mobile phase eluted gradiently at 0.3 mL·min^(-1).The multiple reaction monitoring(MRM)in positive electrospray mode was employed for detection and quantification of the androgens and progestogens.The whole analysis time was 5 mins.Results:The calibration curves of five androgens and progestogens in human serum showed good linearity over the range of 0.05-100 ng·mL^(-1) with a lower limit of quantification at 0.05 ng·mL^(-1).The intra-and inter-day accuracy and precision met the requirements.The variation coefficients of average extraction recovery and internal standard normalized matrix factor were less than 15%.Conclusions:This method can be applied to the determination of 5 sex hormones in clinical serum samples.