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Simultaneous quantitation of endogenous estrone, 17β-estradiol, and estriol in human serum by isotope-dilution liquid chromatography–tandem mass spectrometry for clinical laboratory applications

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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 C18 column 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 E3 was 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-1 for all three estrogens (r 2 > 0.997). The limits of quantification were 5, 10, and 10 pg mL-1 for E1, E2, and E3 respectively, 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 E2 and 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 E2 and unconjugated E3 in serum samples.

Schematic representation of the simultaneous quantitation of three major endogenous estrogens in human serum by ID-LC-MS/MS

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Abbreviations

CLIA:

Chemiluminescence immunoassay

CRM:

Certified reference material

E1 :

Estrone

E1-d 2 :

Estrone-16,16-d 2

E2 :

17β-Estradiol

E2-13 C 3 :

17β-Estradiol-2,3,4-13 C 3

E3 :

Estriol

E3-13 C 3 :

Estriol-2,3,4-13 C 3

16-epE3 :

16-Epiestriol

17-epE3 :

17-Epiestriol

ESI:

Electrospray ionization

ID:

Isotope dilution

IS:

Internal standard

LC:

Liquid chromatography

LOD:

Limit of detection

LOQ:

Limit of quantification

MRM:

Multiple reaction monitoring

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

uE3 :

Unconjugated estriol

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Acknowledgements

We gratefully appreciate the financial support from the National Natural Science Foundation of China (grants 81572088 and 81601861), the Natural Science Foundation of Guangdong Province (grant 2015A030313340), and the Special Translational Medicine of Traditional Chinese Medicine of Guangdong Provincial Hospital (grant 2014KT1593).

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Authors and Affiliations

  1. 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

    Qiaoxuan Zhang, Liqiao Han, Jianbing Wang, Haibiao Lin, Peifeng Ke, Junhua Zhuang & Xianzhang Huang

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  1. Qiaoxuan Zhang
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Correspondence to Junhua Zhuang or Xianzhang Huang.

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The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

The research involving serum samples taken from pregnant women and used in this study was approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (also known as the Second Affiliated Hospital of Guangzhou University of Chinese Medicine) with approval number B2016-159-01.

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Zhang, Q., Han, L., Wang, J. et al. Simultaneous quantitation of endogenous estrone, 17β-estradiol, and estriol in human serum by isotope-dilution liquid chromatography–tandem mass spectrometry for clinical laboratory applications. Anal Bioanal Chem 409, 2627–2638 (2017). https://doi.org/10.1007/s00216-017-0207-z

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  • DOI: https://doi.org/10.1007/s00216-017-0207-z

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