Analytical and Bioanalytical Chemistry

, Volume 411, Issue 27, pp 7095–7104 | Cite as

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography–tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma

  • Qiaoxuan Zhang
  • Lu Zhang
  • Haibiao Lin
  • Zhiliang Cai
  • Jun Yan
  • Qiqin Wang
  • Liqiao Han
  • Jianbing Wang
  • Peifeng Ke
  • Junhua ZhuangEmail author
  • Xianzhang HuangEmail author
Research Paper


A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography–tandem mass spectrometry (ID–LC–MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid–liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID–LC–MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements.

Graphical abstract

Selected ion chromatograms obtained by liquid chromatography–tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample


17α-Hydroxyprogesterone Reference measurement procedure Tandem mass spectrometry Clinical laboratory measurements 





Congenital adrenal hyperplasia


Clinical and Laboratory Standards Institute


Candidate reference measurement procedure


Coefficient of variation


International Federation of Clinical Chemistry and Laboratory Medicine


International Organization for Standardization


Joint Committee for Traceability in Laboratory Medicine


Liquid chromatography


Lower limit of the measuring interval


Limit of detection


Mass spectrometry


Tandem mass spectrometry


International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine


Referenzinstitut für Bioanalytik


Reference measurement procedure



This work was financially supported by the National Key Research and Development Program of China (2017YFF0205401), the National Natural Science Foundation of China (81572088), the Natural Science Foundation of Guangdong Province (2018A0303130124), the Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine (YN2016QJ15 and YN2019QL01), and the Guangzhou Science and Technology Project (201704020213).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Research involving humans and/or animals

The plasma samples were taken from pregnant women and this study is approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine. The number of this review is B2016-159-01.

Informed consent

The Ethics Committee approved the exemption of informed consent.

Supplementary material

216_2019_2086_MOESM1_ESM.pdf (486 kb)
ESM 1 (PDF 485 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Qiaoxuan Zhang
    • 1
    • 2
  • Lu Zhang
    • 2
  • Haibiao Lin
    • 1
    • 2
  • Zhiliang Cai
    • 2
  • Jun Yan
    • 1
  • Qiqin Wang
    • 3
  • Liqiao Han
    • 1
  • Jianbing Wang
    • 1
  • Peifeng Ke
    • 1
  • Junhua Zhuang
    • 1
    Email author
  • Xianzhang Huang
    • 1
    Email author
  1. 1.Department of Laboratory MedicineThe Second Affiliated Hospital of Guangzhou University of Chinese MedicineGuangzhouChina
  2. 2.Second Clinical Medical CollegeGuangzhou University of Chinese MedicineGuangzhouChina
  3. 3.Department of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug ResearchJinan UniversityGuangzhouChina

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