柱前衍生化-超高效液相色谱-三重四极杆串联质谱法测定人血清中11种雌激素的方法学研究

Methodology Study on the Determination of 11 Estrogens in Human Serum by Pre-column Derivatization-UPLC-Triple Quadrupole Tandem Mass Spectrometry

目的建立一种快速、高效、灵敏度高、稳定可靠的柱前衍生化-超高效液相色谱-串联质谱(UPLC-MS/MS)方法,检测人血清中包括雌酮(E1)、雌二醇(E2)、雌三醇(E3)、2-羟基雌酮(2-OHE1)、16α-羟基雌酮(16α-OHE1)等在内的11种超微量雌激素的浓度。方法采用叔丁基甲醚液-液萃取法,并通过丹磺酰氯对萃取后样品进行衍生化处理,以同位素标记的雌酮-13C作为内标,采用Waters ACQUITY UPLC BEH C18色谱柱(2.1 mm×50 mm,1.7μm),0.1%甲酸水-乙腈为流动相,以0.3 mL·min^-1流速等度洗脱,柱温40℃,进样量5μL,在电喷雾离子源(ESI)正离子模式下扫描,多重反应监测(MRM)下测定血清中各类雌激素浓度,分析时间5 min。结果丹磺酰氯衍生化法可大幅度提高各种雌激素在ESI源下的离子化效率,血清中11种雌激素的线性范围为20~2000 pg·mL^-1,定量下限均到达20 pg·mL^-1,提取回收率均可达到70%,批内、批间精密度与准确度均在15%以内,血清样本室温放置24 h、反复冻融3次、-4℃进样盘放置24 h、70℃放置1个月均稳定,满足生物样品测定要求。结论该方法分析时间短、灵敏度高、稳定性好、专属性强,可用于临床血清样本中雌激素代谢网络的定量分析。

Objective To establish a rapid,efficient,sensitive,stable,and reliable pre-column derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the simultaneous determination of 11 ultra-trace estrogens in human serum,including estrone(E1),estradiol(E2),estriol(E3),2-hydroxyestrone(2-OHE1),16α-hydroxyestrone(16α-OHE1).Methods Liquid-liquid extraction using tert-butyl methyl ether was selected for the treatment of human serum samples,and then the derivatization process was performed using dansyl chloride.The isotopically labeled estrone-13C was used as an internal standard.The chromatographic analysis was carried out on a Waters ACQUITY UPLC BEH C18 column(2.1 mm×50 mm,1.7μm)using acetonitrile and water containing 0.1%formic acid as the mobile phase.The flow rate was set at 0.3 mL·min^-1 and the column temperature was controlled at 40℃.The multiple reaction monitoring(MRM)in positive electrospray mode was employed for the detection and quantification of various estrogens in serum,and the analysis time was set at 5 min.Results Dansulyl chloride derivatization could significantly improve the ionization efficiency of various estrogens under ESI source.The linear range of 11 estrogens in serum was 20-2000 pg·mL^-1,with a lower limit of quantification at 20 pg·mL^-1.The recovery rate could reach more than 70%.The precision and accuracy within and between batches were all within 15%.The serum samples,which were placed at room temperature for 24 hours,or were repeatedly frozen and thawed for three times,or were placed in sample tray at-4℃for 24 hours,or were placed in 70℃for 1 month,were stable.The stability met the requirements for biological sample determination.Conclusion This method has the advantages of short analysis time,high sensitivity,good stability and strong specificity.It can be used for the quantitative analysis of estrogen metabolism network in clinical serum samples.