Analytical and Bioanalytical Chemistry

, Volume 411, Issue 28, pp 7519–7528 | Cite as

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

  • Yizhao Chen
  • Hui Ling Teo
  • Hong Liu
  • Tze Ping Loh
  • Qinde LiuEmail author
  • Tang Lin Teo
  • Tong Kooi Lee
  • Sunil Kumar Sethi
Research Paper


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.

Graphical abstract


Testosterone 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


Standard deviation


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

Ethical approval

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.

Supplementary material

216_2019_2152_MOESM1_ESM.pdf (263 kb)
ESM 1 (PDF 262 kb)


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Copyright information

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

Authors and Affiliations

  1. 1.Chemical Metrology Division, Applied Sciences GroupHealth Sciences AuthoritySingaporeSingapore
  2. 2.Department of Laboratory MedicineNational University HospitalSingaporeSingapore

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