Home Medicine Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods
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Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods

  • Qichen Long , Tianjiao Zhang , Ying Yan , Haijian Zhao , Weiyan Zhou , Jie Zeng , Shuijun Li , Jiangtao Zhang , Qingzhang Zeng , Beibei Zhao , Chuanbao Zhang EMAIL logo and Wenxiang Chen EMAIL logo
Published/Copyright: November 25, 2020
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 59 Issue 3

Abstract

Objectives

Accurate measurements of serum 17-hydroxyprogesterone (17OHP) are essential for diagnosis and treatment monitoring for congenital adrenal hyperplasia patients. The performance of serum 17OHP routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of serum 17OHP measurements.

Methods

Serum samples spiked with internal standards were extracted with a combination of solid-phase extraction and liquid-liquid extraction. The 17OHP was quantified by the isotope dilution coupled with liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) with electrospray ionization in positive ion mode. Nine structural analogs of 17OHP were evaluated for interferences. The precision and analytical recovery were assessed. Twenty native and 40 spiked serum for performance evaluation were measured by the cRMP and two clinical LC/MS routine methods.

Results

No apparent interferences were found with the 17OHP measurement. The within-run, between-run, and total precision for our method were 0.4–0.8%, 0.6–2.0%, and 1.0–2.1% for four pooled serum (2.46–102.72 nmol/L), respectively. The recoveries of added 17OHP were 100.0–100.2%. For the performance of two LC/MS routine methods, they showed relative deviation ranges of −22.1 to 1.1% and −6.7 to 12.8%, respectively.

Conclusions

We developed and validated a reliable serum 17OHP method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for serum 17OHP routine measurements.

Keywords: congenital adrenal hyperplasia; liquid chromatography/tandem mass spectrometry; method evaluation; reference measurement procedure; serum 17-hydroxyprogesterone
Corresponding authors: Chuanbao ZhangandWenxiang Chen, National Center for Clinical Laboratories, Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Dahua Road, Dongcheng District, Beijing100730, P.R. China, Phone: +86 010-58115059, Fax: +86 010-65132968, E-mail: (C. Zhang), (W. Chen)

Funding source: The National Key Research and Development Program of China

Award Identifier / Grant number: 2018YFC1002204

Acknowledgments

The authors gratefully acknowledge the colleagues at Beijing Hospital Department of Laboratory Medicine for kind assistance in providing residual patient samples.

  1. Research funding: This research was supported by The National Key Research and Development Program of China (2018YFC1002204).

  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: This study was approved for the exemption from informed consent.

  5. Ethical approval: The study was approved by the Ethics Committee of Beijing Hospital.

References

1. Rauh, M. Steroid measurement with LC-MS/MS in pediatric endocrinology. Mol Cell Endocrinol 2009;301:272–81. https://doi.org/10.1016/j.mce.2008.10.007.Search in Google Scholar PubMed

2. Speiser, PW, Arlt, W, Auchus, RJ, Baskin, LS, Conway, GS, Merke, DP, et al.. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2018;103:4043–88. https://doi.org/10.1210/jc.2018-01865.Search in Google Scholar PubMed PubMed Central

3. Speiser, PW, Azziz, R, Baskin, LS, Ghizzoni, L, Hensle, TW, Merke, DP, et al.. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2010;95:4133–60. https://doi.org/10.1210/jc.2009-2631.Search in Google Scholar PubMed PubMed Central

4. Janzen, N, Riepe, FG, Peter, M, Sander, S, Steuerwald, U, Korsch, E, et al.. Neonatal screening: identification of children with 11beta-hydroxylase deficiency by second-tier testing. Horm Res Paediatr 2012;77:195–9. https://doi.org/10.1159/000337974.Search in Google Scholar PubMed

5. Kulle, A, Krone, N, Holterhus, PM, Schuler, G, Greaves, RF, Juul, A, et al.. Steroid hormone analysis in diagnosis and treatment of DSD: position paper of EU COST Action BM 1303 ‘DSDnet’. Eur J Endocrinol 2017;176:P1–9. https://doi.org/10.1530/eje-16-0953.Search in Google Scholar

6. White, PC. Neonatal screening for congenital adrenal hyperplasia. Nat Rev Endocrinol 2009;5:490–8. https://doi.org/10.1038/nrendo.2009.148.Search in Google Scholar PubMed

7. Turpeinen, U, Itkonen, O, Ahola, L, Stenman, UH. Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay. Scand J Clin Lab Invest 2005;65:3–12. https://doi.org/10.1080/00365510410008421.Search in Google Scholar PubMed

8. Etter, ML, Eichhorst, J, Lehotay, DC. Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method. J Chromatogr B Analyt Technol Biomed Life Sci 2006;840:69–74. https://doi.org/10.1016/j.jchromb.2006.04.038.Search in Google Scholar PubMed

9. Kushnir, MM, Rockwood, AL, Roberts, WL, Pattison, EG, Owen, WE, Bunker, AM, et al.. Development and performance evaluation of a tandem mass spectrometry assay for 4 adrenal steroids. Clin Chem 2006;52:1559–67. https://doi.org/10.1373/clinchem.2006.068445.Search in Google Scholar PubMed

10. Rauh, M, Groschl, M, Rascher, W, Dorr, HG. Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction. Steroids 2006;71:450–8. https://doi.org/10.1016/j.steroids.2006.01.015.Search in Google Scholar PubMed

11. Soeborg, T, Frederiksen, H, Fruekilde, P, Johannsen, TH, Juul, A, Andersson, AM. Serum concentrations of DHEA, DHEAS, 17alpha-hydroxyprogesterone, Delta4-androstenedione and testosterone in children determined by TurboFlow-LC-MS/MS. Clin Chim Acta 2013;419:95–101. https://doi.org/10.1016/j.cca.2013.01.019.Search in Google Scholar PubMed

12. Gervasoni, J, Schiattarella, A, Primiano, A, D’Addurno, I, Cocci, A, Zuppi, C, et al.. Simultaneous quantification of 17-hydroxyprogesterone, androstenedione, testosterone and cortisol in human serum by LC-MS/MS using TurboFlow online sample extraction. Clin Biochem 2016;49:998–1003. https://doi.org/10.1016/j.clinbiochem.2016.05.012.Search in Google Scholar PubMed

13. Greaves, RF, Ho, CS, Loh, TP, Chai, JH, Jolly, L, Graham, P, et al.. Current state and recommendations for harmonization of serum/plasma 17-hydroxyprogesterone mass spectrometry methods. Clin Chem Lab Med 2018;56:1685–97. https://doi.org/10.1515/cclm-2017-1039.Search in Google Scholar PubMed

14. Joint Committee for Traceability in Laboratory Medicine. Available from: https://www.bipm.org/en/committees/jc/jctlm/ [Accessed 10 Sep 2020].Search in Google Scholar

15. Wudy, SA, Hartmann, M, Svoboda, M. Determination of 17-hydroxyprogesterone in plasma by stable isotope dilution/benchtop liquid chromatography-tandem mass spectrometry. Horm Res 2000;53:68–71. https://doi.org/10.1159/000023516.Search in Google Scholar PubMed

16. Tai, SS, Xu, B, Welch, MJ, Phinney, KW. Development and evaluation of a candidate reference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem 2007;388:1087–94. https://doi.org/10.1007/s00216-007-1355-3.Search in Google Scholar PubMed

17. Wang, Y, Zhang, T, Zhao, H, Zhou, W, Zeng, J, Zhang, J, et al.. Measurement of serum progesterone by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method and its application to evaluating immunoassays. Anal Bioanal Chem 2019;411:2363–71. https://doi.org/10.1007/s00216-019-01676-7.Search in Google Scholar PubMed

18. Zhang, T, Zhang, C, Zhao, H, Zeng, J, Zhang, J, Zhou, W, et al.. Determination of serum glucose by isotope dilution liquid chromatography-tandem mass spectrometry: a candidate reference measurement procedure. Anal Bioanal Chem 2016;408:7403–11. https://doi.org/10.1007/s00216-016-9817-0.Search in Google Scholar PubMed

19. Zhang, Q, Han, L, Wang, J, Lin, H, Ke, P, Zhuang, 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 2017;409:2627–38. https://doi.org/10.1007/s00216-017-0207-z.Search in Google Scholar PubMed

20. CLSI. Liquid chromatography-mass spectrometry methods; approved guideline, 1st ed. CLSI document C62-A. Wayne, PA: Clinical Laboratory Standards Institute; 2014.Search in Google Scholar

21. Matuszewski, BK, Constanzer, ML, Chavez-Eng, CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003;75:3019–30. https://doi.org/10.1021/ac020361s.Search in Google Scholar PubMed

22. CLSI. User verification of precision and estimation of bias; approved guideline, 3rd ed. CLSI document EP15-A3. Wayne, PA: Clinical Laboratory Standards Institute; 2014.Search in Google Scholar

23. ISO/IEC Guide 98-3:2008. Uncertainty of measurement – part 3: guide to the expression of uncertainty in measurement (GUM: 1995). Geneva: ISO; 2008.Search in Google Scholar

24. Ellison, SLR, Rosslein, M, Williams, A, editors. EURACHEM/CITAC guide CG 4: quantifying uncertainty in analytical measurement, 2nd ed. Czech Republic: EURACHEM; 2000.Search in Google Scholar

25. RELA-IFCC. External quality control for reference laboratories. Available from: http://dgkl-rfb.de:81/ [Accessed 10 Sep 2020].Search in Google Scholar

26. ISO15193:2009. In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin – Requirements for content and presentation of reference measurement procedures. Geneva: ISO; 2009.Search in Google Scholar

27. Fiet, J, Le Bouc, Y, Guéchot, J, Hélin, N, Maubert, M-A, Farabos, D, et al.. A liquid chromatography/tandem mass spectometry profile of 16 serum steroids, including 21-deoxycortisol and 21-deoxycorticosterone, for management of congenital adrenal hyperplasia. J Endocr Soc 2017;1:186–201. https://doi.org/10.1210/js.2016-1048.Search in Google Scholar PubMed PubMed Central

28. Botelho, JC, Ribera, A, Cooper, HC, Vesper, HW. Evaluation of an isotope dilution HPLC tandem mass spectrometry candidate reference measurement procedure for total 17-β estradiol in human serum. Anal Chem 2016;88:11123–9. https://doi.org/10.1021/acs.analchem.6b03220.Search in Google Scholar PubMed PubMed Central

29. Stockl, D, Sluss, PM, Thienpont, LM. Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis. Clin Chim Acta 2009;408:8–13. https://doi.org/10.1016/j.cca.2009.06.027.Search in Google Scholar PubMed

30. Thienpont, LM. Quality specifications for reference methods. Scand J Clin Lab Invest 1999;59:535–8. https://doi.org/10.1080/00365519950185300.Search in Google Scholar PubMed

31. Westgard. Desirable specifications for total error, imprecision, and bias, derived from intra- and inter-individual biologic variation. Available from: http://westgard.com/biodatabase-2014-update.htm [Accessed 10 Sep 2020].Search in Google Scholar

32. The Royal College of Pathologists of Australasia Quality Assurance Programs. Available from: https://rcpaqap.com.au/ [Accessed 10 Sep 2020].Search in Google Scholar

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-0410).

Received: 2020-03-25
Accepted: 2020-11-02
Published Online: 2020-11-25
Published in Print: 2021-02-23

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2020-0410
Keywords for this article
congenital adrenal hyperplasia; liquid chromatography/tandem mass spectrometry; method evaluation; reference measurement procedure; serum 17-hydroxyprogesterone

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Complex analytical procedures in diagnostic laboratories and the IVDR
  4. Review
  5. Re-examining ferritin-bound iron: current and developing clinical tools
  6. Opinion Papers
  7. Designing a diagnostic Total Testing Process as a base for supporting diagnostic stewardship
  8. The end of the laboratory developed test as we know it? Recommendations from a national multidisciplinary taskforce of laboratory specialists on the interpretation of the IVDR and its complications
  9. EFLM Papers
  10. The Academy of the European Federation of Clinical Chemistry and Laboratory Medicine and the European Register of Specialists in Laboratory Medicine: guide to the Academy and the Register, version 4 – 2020
  11. A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications)
  12. Guidelines and Recommendations
  13. High-sensitivity cardiac troponin I and T methods for the early detection of myocardial injury in patients on chemotherapy
  14. General Clinical Chemistry and Laboratory Medicine
  15. Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods
  16. Evaluation of carbohydrate-deficient transferrin measurements on the V8 capillary electrophoresis system and comparison with the IFCC approved HPLC reference method and N-Latex immunonephelometric assay
  17. The clinical relevance of anti-dsDNA antibodies determined by the Elia™ dsDNA assay in patients with negative indirect immunofluorescence on the HEp-2 cell
  18. A hierarchical bivariate meta-analysis of diagnostic test accuracy to provide direct comparisons of immunoassays vs. indirect immunofluorescence for initial screening of connective tissue diseases
  19. Individual uromodulin serum concentration is independent of glomerular filtration rate in healthy kidney donors
  20. Comparison of iohexol plasma clearance formulas vs. inulin urinary clearance for measuring glomerular filtration rate
  21. Overweight-obesity is associated with decreased vitamin K2 levels in hemodialysis patients
  22. Cardiovascular Diseases
  23. Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin
  24. Infectious Diseases
  25. The anti-inflammatory cytokine response characterized by elevated interleukin-10 is a stronger predictor of severe disease and poor outcomes than the pro-inflammatory cytokine response in coronavirus disease 2019 (COVID-19)
  26. Quantification of the HIV-1 total reservoir in the peripheral blood of naïve and treated patients by a standardised method derived from a commercial HIV-1 RNA quantification assay
  27. Comparison of the reliability of Gram-negative and Gram-positive flags of the Sysmex UF-5000 with manual Gram stain and urine culture results
  28. Evaluation of a turbidimetric C-reactive protein assay to monitor early-onset neonatal sepsis in South Kivu (Democratic Republic of the Congo)
  29. Corrigendum
  30. Corrigendum to: Procalcitonin (PCT)-guided antibiotic stewardship in Asia-Pacific countries: adaptation based on an expert consensus meeting
  31. Letters to the Editor
  32. Decision-making based on sFlt-1/PlGF ratios: are immunoassay results interchangeable for diagnosis or prognosis of preeclampsia?
  33. Inter-center agreement in the interpretation of oligoclonal bands
  34. A new founder BRCA1 haplotype identified in the Puglia region is associated with a specific age-related cancer onset in three unrelated families
  35. Predictive values of D-dimer for adverse pregnancy outcomes: a retrospective study
  36. A very uncommon haemoglobin value resulting from a severe acute malnutrition in a 16-month-old child in Ethiopia
  37. Ionized calcium measurements during continuous renal replacement therapy with regional citrate anticoagulation
  38. The hairy cell leukaemia oxymoron: monocytotic monocytopenia
  39. Perchlorate interference with electrolyte analysis
  40. False decrease in serum triglyceride and cholesterol due to massive ascorbate in a bowel preparation solution
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