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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 pretreatment method of serum free estriol (uE3) by high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). Methods The activated carbon adsorbed sera was used as the research matrix. The LC-20AD XR high performance liquid chromatography system and the API 5500 The tandem quadrupole mass spectrometer was optimized for the E3 detection of chromatographic conditions, mass spectrometry conditions and extraction conditions. Results The chromatographic conditions: Phenomenex Knietex column (100.0mm × 2.1mm, 2.6μm) was used. The organic phase was methanol and the aqueous phase was 0.1mmol / L ammonium fluoride aqueous solution with 8min gradient. Mass spectrometry conditions: ESI-MS mode and MRM mode analysis were used to select m / z 287 → m / z 145 as the quantitative ion pair of E3, m / z 287 → m / z 171 was used as the qualitative monitoring ion pair. Quantitative ion pairs with m / z 290 → m / z 148 as the internal standard and m / z 290 → m / z 174 as the ion pair for qualitative monitoring were selected. Extraction conditions: Extractant is n-hexane / ethyl acetate (50/50, v / v), sample and extractant ratio of 1: 2, extraction rate of up to 85.71%. Conclusion The chromatographic conditions, mass spectrometry conditions and extraction conditions of E3 detection have been fully optimized, which laid a good foundation for the subsequent establishment of a reference method for the determination of uE3 in human blood by LC-MS / MS.