同位素稀释-超高效液相色谱串联质谱法检测血浆中的皮质醇

Isotope Dilution- Ultra Performance Liquid Chromatography Tandem Mass Spectrometry Method for the Detection of Plasma Cortisol

目的：建立一种同位素稀释-超高效液相色谱串联质谱法检测血浆中的皮质醇。方法：使用C18固相抽提小柱纯化、富集人体血浆中的总皮质醇。采用LC/MS/MS-ESI法分析血浆中的皮质醇含量。梯度流动相为0 min,50%甲醇;3 min,90%甲醇;3.5 min 90%甲醇;3.6min 50%甲醇;5 min 50%甲醇。水相含0.2%甲酸。MRM检测模式选择性监测cortisol（m/z363.2/121.4）、内标cortisol-d4（m/z367.4/121.4）。结果 ：血浆中加入梯度标准品的加标回收率分别为102.5%,101.1%,103.3%。检测冻存血浆中皮质醇的实验里,组内变异为0.54%-2.47%,组间变异为0.96%-1.69%,线性相关系数为R^2≥0.999。LC/MS/MS信噪比为10时检出限为12pg。结论：该方法具有良好的特异性、灵敏度、精密度,能够准确的检测血浆中总皮质醇含量,为实现皮质醇溯源打下基础。

Objective： To establish a stable isotope dilution- ultra performance liquid chromatography tandem mass spectrometry method for the detection of plasma cortisol. Methods： Total cortisol concentration in human plasma and purification, using C18 solid phase extraction column. Cortisol concentrations in plasma were analyzed by using LC/MS/MS-ESI method. A gradient mobile phase was 0 min, 3 min, 50% methanol, 90% methanol; methanol; 3.5 min 90% 3.6min 50%; 5 min 50% of methanol; methanol. The aqueous phase containing 0.2% formic acid. MRM detection mode selective monitoring of cortisol（m/z363.2/121.4） cortisol-d4（m/z367.4/121.4）, the internal standard. Results： The recovery of added standard gradient in plasma was respectively 102.5%, 101.1%, 103.3%.Detection of plasma cortisol in freezing experiment, within group variation is 0.54% - 2.47%, 0.96% - 1.69% between group variation,linear correlation coefficient is R^2= 0.999. LC/MS/MS signal to noise ratio is 10, the detection limit is 12 pg. Conclusion： The method has good specificity, sensitivity, precision, detection of plasma total cortisol levels can be accurate, lay the foundation for the realization of cortisol.