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Abstract: Abstract：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 mL/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 (LoQ) 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.
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