同位素稀释超高效液相色谱-串联质谱法测定人血清中孕酮

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)测定人血清中孕酮的分析方法。血清样品经乙酸乙酯、正己烷液液萃取(LLE)后,采用Acquity UPLC BEH C18色谱柱(2.1 mm×100 mm,1.7μm)进行梯度分离,色谱运行时间为5 min,采用电喷雾(ESI)正离子电离模式和多反应监测(MRM)扫描模式,同位素内标法定量。考察了两步萃取法对孕酮的提取效果,不同流动相的分离效果以及样品稳定性,结果表明以甲醇-0.1%氨水溶液为流动相时分离效果较好。优化条件下,孕酮在10~10000 pg/mL范围内线性关系良好(r^2=0.9998),方法检出限和定量下限分别为5、10 pg/mL;平均加标回收率为91.5%~106%,日内相对标准偏差(RSD)为1.2%~8.2%,日间RSD为4.1%~9.1%。采用该方法对30个真实血清样品进行测定,孕酮质量浓度为0.0502~1.3635 ng/mL,均在正常生理范围内。该方法灵敏度高、准确可靠,可用于临床血清样品中孕酮生理水平的检测。

Determination of Progesterone in Human Serum Using Isotope Dilution Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

An ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)with isotope dilution was developed for the analysis of progesterone in human serum.The serum samples were extracted by liquid-liquid extraction(LLE)with ethyl acetate and n-hexane,then separated on an Acquity UPLC BEH C18 column(2.1 mm×100 mm,1.7μm)by gradient elution in 5 min,and finally analyzed by UPLC-MS/MS with electrospray ionization in positive ion mode under multiple reaction monitoring(MRM)mode and quantified by isotope internal standard.Impact of the two-step LLE extraction on progesterone was investigated.Higher separation performance was observed when methanol-water containing 0.1%aqueous ammonia was used as mobile phase.The stability of serum extracts was also investigated.Results showed that the calibration curves for progesterone were linear in the concentration range of 10-10000 pg/mL with correlation coefficients(r^2)of 0.9998.The limit of detection(LOD,S/N=3)and limit of quantitation(LOQ,S/N=10)were 5 pg/mL and 10 pg/mL,respectively.Recoveries for progesterone ranged from 91.5%to 106%with the intra-relative standard deviations(RSD)and inter-RSD of 1.2%-8.2%and 4.1%-9.1%,respectively.This method was applied in the determination of progesterone in serum samples from thirty individual donors with the progesterone concentrations ranging from 0.0502 ng/mL to 1.3635 ng/mL.With the characteristics of high sensitivity,good accuracy and reliability,this method could be used in the detection of progesterone in clinical serum samples.