高效液相色谱串联质谱法测定血清非结合雌三醇的前处理研究

Investigate the pretreat conditions of the determination of unconjugated estriol in human serum using high performance liquid chromatography tandem mass spectrometry

目的优化高效液相色谱串联质谱法(LC-MS/MS)测定血清游离雌三醇(uE3)的前处理方法。方法以活性炭吸附血清为研究基质,通过加入已知量的雌三醇(E3)标准品及E3-2-3-4-13 C3内标,在LC-20AD XR高效液相色谱系统和API 5500串联四级杆质谱仪上对E3检测的色谱条件、质谱条件及萃取条件等进行优化。结果色谱条件:选用菲罗门Knietex色谱柱(100.0mm×2.1mm,2.6μm);有机相为甲醇,水相为0.1mmol/L氟化铵水溶液,8min梯度洗脱。质谱条件:选用电喷雾(ESI)负离子模式和多反应监测(MRM)模式分析,选择质荷比(m/z)287→m/z 145作为E3的定量离子对,m/z 287→m/z 171作为定性监测离子对;选择m/z 290→m/z 148作为内标的定量离子对,m/z 290→m/z 174作为定性监测的离子对。萃取条件:萃取剂为正己烷/乙酸乙酯(50/50,v/v),样品与萃取剂的比例为1∶2,萃取率可达85.71%。结论 E3检测的色谱条件、质谱条件、萃取条件已得到全面优化,为后续建立LC-MS/MS测定人血中uE3的参考方法打下良好基础。

Objective To optimize the pretreat conditions of the determination of unconjugated estriol（uE3）in human serum using isotope dilution liquid chromatography/tandem mass spectrometry（ID-LC-MS/MS）.MethodsEstriol（purity99.3% ）and estriol-2-3-4-13 C3 were the standard and the internal standard respectively.Activated carbon adsorption serum was used for the study of serum matrix.The liquid chromatography（LC）and mass spectrometry（MS）condition were optimized to increase the sensitivity of the detection by use of the the AB/Sciex API 5500 tandem mass spectrometer equipped with Shimadzu LC-20 AD XR system.Results LC condition：Knietex 100×2.1mm,2.6μm as the column,the mobile phase was fluoride（NH4F）in water（buffer A）and methanol（buffer B）.The analysis of uE3 was accomplished within 8min.MS condition：the samples were analyzed by ID-LC-MS/MS system in the negativelyelectro-spray ioniz ation（ESI）mode and multiple reaction monitor（MRM）mode.And the MRM transitions used for each analytes were：（m/z）287→145（quantitative）and m/z 287→171（qualitative）for uE3,m/z 290→148（quantitative）and m/z 290→174（qualitative）for internal standard.Extraction：n-Hexane/ethyl acetate（50∶50,v/v）was chosen to the extraction solvent,and the volume ratio of the serum sample to extraction solvent was 1∶2.The mean extraction efficiency for the method was determined to be 85.71% .Conclusion This study successfully optimized the LC and MS conditions and the ideal condition was obtained of the determination of uE3 in human serum using ID-LC-MS/MS.The applicable extract method was determined.This study was an important base of establish the candidate reference measurement procedure for the determination of uE3 in human serum using ID-LC-MS/MS.