液相色谱-串联质谱法检测血清中15种甾体激素的动态变化

建立了用液相色谱-串联质谱检测血清中15种甾体激素的方法。在血清样品中加入含内标的乙腈沉淀蛋白质后,用100 mmol/L盐酸羟胺衍生,经Agilent-C18柱(50 mm×3.0 mm,2.7μm)分离,内标法定量。质谱分析采用电喷雾电离(ESI)正离子扫描,多反应监测(MRM)模式检测。数据显示,13种激素在0.05~20 ng/mL范围内线性关系良好(相关系数(R2)不小于0.995 6),检出限不大于1 ng/mL;皮质醇及脱氢表雄酮硫酸酯在50~2 000ng/mL范围内线性关系良好(R2不小于0.997 9),检出限不大于0.5 ng/mL。将该方法应用于女性月经周期激素变化规律的研究,结果表明,15种甾体激素在一个完整的月经周期内有不同特征的动态变化,其中雄激素在滤泡期的后期达到最高值,孕(雌)激素在黄体期达到最高值,而皮质激素则波动不明显。该方法灵敏度高、重复性好,为临床诊疗提供了参考。

Monitoring of dynamic changes of 15 steroids in serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of 15 steroids. Acetonitrile containing internal standard was added to the serum samples to precipitate the proteins. Then the derivatization of the samples was conducted with 100 mmol/L hydroxylamine hydrochloride. The separation of the samples was performed on an Agilent-C18 column (50 mm×3.0 mm, 2.7 μm), and quantified with internal standard method. The steroids were determined by multiple-reaction monitoring (MRM) with electrospray ionization (positive mode) MS. The 13 steroids showed linear relationships from 0.05 to 20 ng/mL (R²≥0.9956) with limits of detection no more than 1 ng/mL; cortisol and dehydroepiandrosterone sulfate showed linear relationships from 50 to 2000 ng/mL (R²≥0.9979) with limits of detection less than 0.5 ng/mL. The method was used for the monitoring of the dynamic changes of steroids in a menstrual cycle of women. The 15 steroids fluctuated with different properties, in which androgens reached the peak in late follicular phase, progesterone (estrogens) reached the peak in luteal phase, while the changes of cortical steroids were not so evident. The method is sensitive and repeatable. The data in this project can provide some references for clinical practice.