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Influence of isotopically labeled internal standards on quantification of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography mass spectrometry

  • Tze Ping Loh , Chung Shun Ho , Michaela F. Hartmann , Rosita Zakaria , Clara Wai Shan Lo , Sjoerd van den Berg , Yolanda B. de Rijke , Brian R. Cooke , Kirsten Hoad , Peter Graham , Stephen R. Davies , Lindsey G. Mackay , Stefan A. Wudy and Ronda F. Greaves ORCID logo EMAIL logo



Our recent survey of 44 mass spectrometry laboratories across 17 countries identified variation in internal standard (IS) choice for the measurement of serum/plasma 17α-hydroxyprogesterone (17OHP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The choice of IS may contribute to inter-method variations. This study evaluated the effect of two common isotopically labeled IS on the quantification of 17OHP by LC-MS/MS.


Three collaborating LC-MS/MS laboratories from Asia, Europe and Australia, who routinely measure serum 17OHP, compared two IS, (1) IsoSciences carbon-13 labeled 17OHP-[2,3,4-13C3], and (2) IsoSciences deuterated 17OHP-[2,2,4,6,6,21,21,21-2H]. This was performed as part of their routine patient runs using their respective laboratory standard operating procedure.


The three laboratories measured 99, 89, 95 independent samples, respectively (up to 100 nmol/L) using the 13C- and 2H-labeled IS. The slopes of the Passing-Bablok regression ranged 0.98–1.00 (all 95% confidence interval [CI] estimates included the line of identity), and intercept of <0.1 nmol/L. Average percentage differences of −0.04% to −5.4% were observed between the two IS materials, which were less than the optimal bias specification of 7% determined by biological variation, indicating no clinically significant difference. The results of 12 Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) proficiency samples (1–40 nmol/L) measured by the laboratories were all within the RCPAQAP analytical performance specifications for both IS.


Overall, the comparison between the results of 13C- and 2H-labeled IS for 17OHP showed good agreement, and show no clinically significant bias when incorporated into the LC-MS/MS methods employed in the collaborating laboratories.

Corresponding author: Ronda F. Greaves, Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville, Victoria, Australia; School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia; and Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, E-mail:

Funding source: National University of Singapore

Award Identifier / Grant number: Unassigned


We are grateful for the support of Mr. Michael Rennie (PM Separations, Queensland, Australia) for facilitating the purchase and distribution of the Isosciences Internal Standards worldwide. We also gratefully acknowledge the funding provided by the BIGHEART Seed Grant (National University of Singapore) to TPL that enabled the procurement and distribution of the materials. This work was part of a collaborative project under the umbrella of the Asia Pacific Federation of Clinical Biochemistry and Laboratory Medicine (APFCB) Mass Spectrometry Harmonisation Working Group, with participation from: (1) the Australasian Association of Clinical Biochemistry and Laboratory Medicine (AACB); (2) National Measurement Institute of Australia (NMIA); (3) Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP); (4) the International Federation of Clinical Chemistry and Laboratory Medicine’s Committee for Emerging Technologies in Pediatric Laboratory Medicine (IFCC C-ETPLM); and (5) the individual laboratories conducting the experiments.

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The local Institutional Review Board deemed the study exempt from review.


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Received: 2020-03-15
Accepted: 2020-06-02
Published Online: 2020-07-22
Published in Print: 2020-09-25

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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