title: Simultaneous quantitation of endogenous estrone, 17β-estradiol, and estriol in human serum by isotope-dilution liquid chromatography-tandem mass spectrometry for clinical laboratory applications.
abstract: Estrogen measurements are important in the assessment of female reproductive function and have expanding roles in other fields. A simple, accurate, highly sensitive and specific isotope-dilution liquid chromatography-tandem mass spectrometry method was developed and evaluated to simultaneously measure three endogenous estrogens in serum: estrone (E1), 17β-estradiol (E2), and estriol (E3). Chromatographic separation was achieved on a C18column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode. The sample preparation in this assay requires no derivatization and extraction by liquid-liquid extraction. After optimization of the extraction conditions, the final extraction efficiency of E1, E2, and E3was 83.8%, 78.9%, and 77.3% respectively. The metabolites and structural analogs that have the same molecular masses as the estrogens were separated under the optimized liquid chromatography conditions. Method validation showed satisfactory linearity over the concentration range of 20-10000 pg mL-1for all three estrogens (r2> 0.997). The limits of quantification were 5, 10, and 10 pg mL-1for E1, E2, and E3respectively, and their recoveries ranged from 94.7% to 103.5%. The accuracy of the proposed method was further evaluated with use of certified reference materials BCR-576, BCR-577, and BCR-578 for E2and 2014 International Federation of Clinical Chemistry and Laboratory Medicine External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine samples for E3, whose certified values were determined by reference methods. Great agreement was observed between the measured values and the certified values. Satisfactory precision (coefficients of variation less than 7.44%) was also obtained for the three estrogens. Moreover, the proposed method was successfully applied to measure the three estrogens in serum samples of pregnant women in the second trimester and to assess the accuracy of chemiluminescent immunoassays in clinical laboratories by determination of E2and unconjugated E3in serum samples. Graphical Abstract Schematic representation of the simultaneous quantitation of three major endogenous estrogens in human serum by ID-LC-MS/MS.
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China.
authors.forename: Qiaoxuan
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China.
authors.forename: Liqiao
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China.
authors.forename: Jianbing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China.
authors.forename: Haibiao
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China.
authors.forename: Peifeng
authors.initials: P
authors.lastname: Ke
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China. zjh2208@163.com.
authors.forename: Junhua
authors.initials: J
authors.lastname: Zhuang
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), No. 111, Dade Road, Yuexiu District, Guangzhou, Guangdong, 510120, China. huangxz020@163.com.
authors.forename: Xianzhang
authors.initials: X
authors.lastname: Huang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D019411:Clinical Laboratory Techniques / Q000592:standards*; D004958:Estradiol / Q000097:blood*; D004964:Estriol / Q000097:blood*; D004970:Estrone / Q000097:blood*; D005260:Female; D006801:Humans; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004970:Estrone; D004958:Estradiol; D004964:Estriol
keywords: Clinical laboratory; Endogenous estrogens; Liquid–liquid extraction; Mass spectrometry; Serum samples