Analytical and Bioanalytical Chemistry

, Volume 410, Issue 24, pp 6257–6267 | Cite as

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

  • Xianzhang Huang
  • Qiaoxuan Zhang
  • Songbai Zheng
  • Jianbing Wang
  • Liqiao Han
  • Haibiao Lin
  • Peifeng Ke
  • Junhua ZhuangEmail author
  • Zhimin (Tim) CaoEmail author
Research Paper


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.

Graphical abstract

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


Estriol Reference method Tandem mass spectrometry Immunoassay 



unconjugated estriol


liquid chromatography/tandem mass spectrometry


multiple reaction monitoring


reference measurement procedure


Joint Committee for Traceability in Laboratory Medicine


proficiency testing


Reference Laboratories in Laboratory Medicine


International Federation of Clinical Chemistry and Laboratory Medicine



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).

Compliance with ethical standards

Conflicts of interest

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.

Informed consent

Ethics committee approved the exemption of informed consent.

Supplementary material

216_2018_1236_MOESM1_ESM.pdf (16 kb)
ESM 1 (PDF 16 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Xianzhang Huang
    • 1
  • Qiaoxuan Zhang
    • 1
  • Songbai Zheng
    • 1
  • Jianbing Wang
    • 1
  • Liqiao Han
    • 1
  • Haibiao Lin
    • 1
  • Peifeng Ke
    • 1
  • Junhua Zhuang
    • 1
    Email author
  • Zhimin (Tim) Cao
    • 2
    • 3
    Email author
  1. 1.Department of Laboratory MedicineThe Second Affiliated Hospital of Guangzhou University of Chinese MedicineGuangzhouChina
  2. 2.Wadsworth CenterNew York State Department of HealthAlbanyUSA
  3. 3.College of Arts and SciencesUniversity at Albany, State University of New YorkAlbanyUSA

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