Simple and accurate candidate reference measurement procedure for total testosterone in human serum by one-step liquid-liquid extraction coupled with isotope dilution mass spectrometry
Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2 h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from −0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future.
KeywordsTestosterone Human serum Reference measurement procedure Isotope dilution mass spectrometry Liquid chromatography-tandem mass spectrometry Liquid-liquid extraction
Certified reference material
Coefficient of variation
Isotope dilution mass spectrometry
Liquid chromatography-tandem mass spectrometry
The International System of Units
The authors are grateful to the Health Sciences Authority, Singapore for the support of this project.
Compliance with ethical standards
The use of leftover patient serum samples in this study has been approved by the National Healthcare Group Domain Specific Review Board (reference no. DSRB 2016/01201) and complied with the Declaration of Helsinki.
Conflict of interest
The authors declare that there is no conflict of interest.
- 10.de Bièvre P, Peiser HS. Basic equations and uncertainties in isotope-dilution mass spectrometry for traceability to SI of values obtained by this primary method. Fresenius J Anal Chem. 1997;359(7–8):523–5.Google Scholar
- 14.van Uytfanghe K, Stöckl D, Kaufman JM, Fiers T, Ross HA, de Leenheer AP, et al. Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry. Clin Chem. 2004;50(11):2101–10.CrossRefGoogle Scholar
- 22.Yong S, Liu H, Wong L, Teo HL, Chen Y, Liu Q, et al. Liquid chromatography-isotope dilution tandem mass spectrometry method for the measurement of urea in human serum and assignment of reference values to external quality assessment samples. Int J Mass Spectrom. 2017;414:87–93.CrossRefGoogle Scholar
- 23.ISO 15193:2009 In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin – Requirements for content and presentations of reference measurement procedures.Google Scholar
- 24.ISO/IEC Guide 98–3:2008 Uncertainty of measurement – Part 3: Guide to the expression of uncertainty in measurement (GUM:1995).Google Scholar
- 25.Jenkison C, Deshmukh NIK, Shah I, Zachár G, Székely AD, Petroczi A, et al. LC-MS/MS-Based assay for free and deconjugated testosterone and epitestosterone in rat urine and serum. J Anal Bioanal Tech. S5:006. https://doi.org/10.4172/2155-9872.S5-006.
- 27.United States Environmental Protection Agency. Chemistry Dashboard. Available at: https://comptox.epa.gov/dashboard/dsstoxdb/results?utf8=%E2%9C%93&search=testosteone. Accessed 8 Aug 2018.
- 28.United States Environmental Protection Agency. Chemistry Dashboard. Available at: https://comptox.epa.gov/dashboard/dsstoxdb/results?utf8=%E2%9C%93&search=Progesterone. Accessed 8 Aug 2018.
- 29.United States Environmental Protection Agency. Chemistry Dashboard. Available at: https://comptox.epa.gov/dashboard/dsstoxdb/results?utf8=%E2%9C%93&search=Cortisol. Accessed 8 Aug 2018.
- 31.2019: CLIA proposed changes to PT acceptable limits. https://www.westgard.com/2019-clia-changes.htm. Accessed 1 Aug 2019.
- 32.Allowable Limits of Performance — RCPAQAP. http://www.rcpaqap.com.au/docs/2014/chempath/ALP.pdf. Accessed 1 Aug 2019.