超快速液相色谱-串联质谱法检测人血清中雌酮、雌二醇及雌三醇的方法学研究

Study on the determination of estrone,estradiol and estriol in human serum by UFLC-MS/MS method with pre-column derivatization

建立灵敏、可靠、稳定的柱前衍生化超快速液相色谱-串联质谱（ultra-fast liquid chromatography-mass spectrometry,UFLC-MS/MS）方法检测人血清中超微量雌酮、雌二醇和雌三醇。方法：取200μL血清样品,采用两步液萃取法,通过丹磺酰氯衍生化后进行样品分析,以雌二醇-d3作为内标。采用Welch Ultimate UHPLC XB-C18色谱柱（2.1mm×50mm,1.8μm）,乙腈-水（含0.1%甲酸）为流动相,梯度洗脱,流速0.20mL/min,柱温40℃,进样量10μL。在电喷雾离子（electrospray ionization,ESI）源正离子模式下,选择多反应监测（multiple reaction monitoring,MRM）模式。经检测,雌酮、雌二醇、雌三醇及内标的检测离子对分别为：m/z为504.0→171.0、m/z为506.0→171.0、m/z为522.0→171.0和m/z为509.0→171.0。结果：丹磺酰氯衍生法可大幅度提高雌酮、雌二醇、雌三醇在ESI源下的离子化效率,血清中3种成分的线性范围均为0.01-5.00μg/L,定量下限均达到10ng/L,样品回收率50%以上,批内、批间准确度与精密度均小于15%,满足生物样品的分析要求。结论：该方法灵敏度高、重现性好,可以用于临床血清样品中雌激素水平的定量分析。

A selective and sensitive ultra-fast liquid chromatography-mass spectrometry （UFLC-MS/MS） method with pre-column derivatization was developed for the simultaneous determination of estrone, estradiol and estriol in human serum. The analytes were extracted from 200 μL serum samples by liquid-liquid extraction using two-step extraction method via methyl tert-butyl ether and dichloromethane. And then derivatization process was carried out using dansyl chloride. Estradiol-d3 was selected as the internal standard. Chromatographic separation was achieved on a Welch Ultimate UHPLCXB-C18 column （2. 1 mm× 50 mm, 1.8 μm） using mobile phase consisted of aeetonitrile and water containing 0. 1% formic acid. The column temperature was 40℃. The flow rate was set at 0. 20 mL/min and the injection volume was 10μL. Detection and quantification were performed using a mass spectrometer by the multiple reaction monitoring （MRM） in positive electrospray ionization mode. The transition monitored were m/z 504. 0→171.0 for estrone, m/z 506. 0→171.0 for estradiol, m/z 522. 0→171.0 for estriol and m/z 509.0→171.0 for IS, respectively. The calibration curves of all three analytes in serum show good linearity over the concentration range of 0. 01-5.00 μg/L with a limit of quantitation at 10 ng/L. The extraction recoveries are 〉50% as well as both the intra-and intevday accuracy and precision are within 15%. The sensitive and reproducible method could be successfully applied for the quantification of estrone, estradiol and estriol in clinical serum samples.