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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
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作者单位
薛晋美,孙光,李会强,那平,乔斌 1.天津大学化工学院2.天津市公安医院检验科3.天津医科大学检验学院 
基金项目:国家自然科学基金项目(81772259)
中文摘要: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,1.7 μm)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10~10 000 pg/mL范围内线性关系良好(r2=0.999 8),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0.050 2~1.363 5 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。
中文关键词:孕酮  同位素稀释  超高效液相色谱-串联质谱(UPLC-MS/MS)  液液萃取(LLE)  血清
 
Determination of Progesterone in Human Serum Using Isotope Dilution Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry
Abstract: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,1.7 μm) 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 10-10 000 pg/mL with correlation coefficients(r2) of 0.999 8.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 0.050 2 ng/mL to 1.363 5 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.
Key Words:progesterone  isotope dilution  ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)  liquid-liquid extraction(LLE)  serum
引用本文:薛晋美,孙光,李会强,那平,乔斌.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[J].分析测试学报,2020,39(5):652-656.
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