液相色谱串联质谱法检测人血清雌酮、雌二醇

Determination of oestrone and estradiol in serum based on liquid chromatography-tandem mass spectrometry

目的建立同时测定人血清中雌酮（E1）及雌二醇（E2）水平的液相色谱串联质谱（LC-MS/MS）法,并初步应用于临床血清样品的雌激素检测。方法血清样品经乙酸乙酯提取,丹磺酰氯衍生化后进行LC-MS/MS分析。用Agilent ZORBAX SBC18色谱柱进行线性梯度洗脱,流动相为乙腈（0.05%甲酸）-水（0.05%甲酸）,流速为0.3 m L/min。用多重反应离子监测（MRM）正离子模式对血清中的E1、E2进行质谱检测,并根据＂生物样品定量分析方法验证指导原则（中国药典,2015）＂对该方法的特异性、线性范围、灵敏度、精密度、提取回收率和稳定性等进行评价。用本法对172例健康成年女性血清标本进行E1、E2检测,用百分位数法计算不同月经周期E1、E2的浓度区间。结果 LC-MS/MS法可同时检测人血清中E1、E2的含量,在0.05~10 nmol/L范围内线性关系良好。该法的定量限为0.05 nmol/L,日内与日间不精密度均小于15%,稳定性良好。初步临床应用显示,用LC-MS/MS法计算所得的E1、E2浓度区间符合女性月经周期生理性变化规律。结论 LC-MS/MS法能有效分离并定量检测人血清中E1、E2的浓度水平,其线性范围广、灵敏度高、精密度好,有望作为临床血清雌激素检测的参考方法。

Objective To establish a high liquid chromatography-tandem mass spectrometry（ LC-MS / MS） method for simultaneous determination of estrone（ E1） and estradiol（ E2） in human serum and determine estrogen level in clinical samples using the established method. Methods Serum samples were extracted by ethyl acetate and derivatized with dansyl chloridefor determination of LC-MS /MS. Linear gradient elution was performed on Agilent ZORBAX SB-C18 column with mobile phase comprising acetonitrile-water（ 0. 05% formic acid） and flow rate of 0. 3 m L/min. The levels of oestrone and estradiol in serum were determined by multiple reaction monitoring（ MRM） mode with positive electrospray ionization（ ESI）. The performance of the methodology,including specificity,linearity range,sensitivity,precision,extraction recovery and stability,was verified according to the Guideline of quantitative analysis validation of biological samples（ Chinese Pharmacopoeia,2015）. The serum samples from 172 healthy adult females were analyzed and the concentration ranges of oestrone and estradiol at different phases of menstrual cycle were calculated with percentile method. Results The established LC-MS / MS method could quantify the levels of oestrone and estradiol in serum simultaneously with fine stability and linearity in the range from 0. 05 to 10 nmol / L. The limit of quantitation（ Lo Q） was 0. 05 nmol / L,and the imprecision of intra-and inter-batch precision was less than 15 percent,The preliminary clinical application showed that the concentration ranges of oestrone and estradiol calculated with LC-MS / MS method met with the physiological rhythms of menstrual cycle in females. Conclusion The established LC-MS / MS method could differentiate and quantify the levels of oestrone and estradiol in serum efficiently,which was characterized with broad analytical range and approving sensitivity and precision. The method should be expected to become a candidate reference method for clinical determination of serum estrogen.