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Measurement of human serum unconjugated estriol without derivatization using liquid chromatography-tandem mass spectrometry candidate reference method and compared with two immunoassays

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Abstract

A candidate reference measurement procedure (RMP) for measurement of unconjugated estriol in human serum has been developed and validated. The proposed method is highly reliable and uses isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and requires no derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotopically labeled internal standard. Unconjugated estriol and its internal standard were extracted from serum matrix using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Calibrator bracketing was used to give higher specificity and accuracy for assigning serum level. The accuracy of the candidate RMP was validated by split-sample comparison to established RMPs. The lowest limit of detection (LLoD) and lowest limit of quantification (LLoQ) for developed RMP was estimated to be 0.14 nmol/L and 0.35 nmol/L, respectively. Both intra- and inter-assay imprecisions were ≤2.19% at 1.39, 17.34 and 69.35 nmol/L, respectively. Recoveries were 98.54% to 100.34% and linear response ranged from 0.35 to 173.38 nmol/L. No interference was observed. Biases were 5.6% and 2.8% against the targets of RELA2015A (3.87 nmol/L) and RELA2015B (40.62 nmol/L), respectively. Moreover, the candidate RMP was successfully applied to measure level of unconjugated estriol in serum samples of pregnant women (n = 3) and compared with two immunoassays in clinical laboratory. Our developed method is simple, accurate, and can be used as a candidate RMP to determine total unconjugated estriol level in human serum. Further improvement of certain immunoassays in accuracy and precision is needed.

Selected ion chromatograms by LC-MS/MS using a C18 column for uE3 from a serum sample

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Abbreviations

uE3 :

unconjugated estriol

LC-MS/MS:

liquid chromatography/tandem mass spectrometry

MRM:

multiple reaction monitoring

RMP:

reference measurement procedure

JCTLM:

Joint Committee for Traceability in Laboratory Medicine

PT:

proficiency testing

RELA:

Reference Laboratories in Laboratory Medicine

IFCC:

International Federation of Clinical Chemistry and Laboratory Medicine

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Acknowledgements

The authors thank Prof. Zhiguo Wang for sharing information on the uE3 results of the PT event 2016 conducted by China National Center for Clinical Laboratory.

Financial support

This work was supported by the National Natural Science Foundation of China (81572088), the Natural Science Foundation of Guangdong Province (2015A030313340), the Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine (YN2014ZHR204 and YN2016QJ15), and Administration of Traditional Chinese Medicine Bureau of Guangdong Province (20172063).

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Authors and Affiliations

  1. Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510120, Guangdong, China

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

  2. Wadsworth Center, New York State Department of Health, Albany, NY, 12201-0509, USA

    Zhimin (Tim) Cao

  3. College of Arts and Sciences, University at Albany, State University of New York, Albany, NY, 12222, USA

    Zhimin (Tim) Cao

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  1. Xianzhang Huang
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Correspondence to Junhua Zhuang or Zhimin (Tim) Cao.

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All authors declare that they have no conflict of interest.

The research involved human participants and/or animals

The research involves serum samples taken from pregnant women and this study is approved by Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (the other name: Second Affiliated Hospital of Guangzhou University of Chinese Medicine). The number of this Review is: B2016-159-01.

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Ethics committee approved the exemption of informed consent.

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The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official views or positions of the Wadsworth Center of New York State Department of Health. Use of trade names and commercial sources is for identification only and does not constitute endorsement by New York State Department of Health.

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Huang, X., Zhang, Q., Zheng, S. et al. Measurement of human serum unconjugated estriol without derivatization using liquid chromatography-tandem mass spectrometry candidate reference method and compared with two immunoassays. Anal Bioanal Chem 410, 6257–6267 (2018). https://doi.org/10.1007/s00216-018-1236-y

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