An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of testosterone in human serum and plasma
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Abstract
Objectives
This study presents a candidate reference measurement procedure (RMP) for testosterone quantification in human serum and plasma, utilizing isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS).
Methods
The developed LC-MS/MS method employs a two-dimensional heart-cut LC approach combined with solid-phase extraction (SPE) for sample clean-up, ensuring accurate testosterone analysis in human serum and plasma. Traceability to SI units was achieved by using a primary reference material listed by the Joint Committee on Traceability in Laboratory Medicine (JCTLM). An alternative quantification approach using qNMR content determination is also described. Assay validation followed current guidelines, assessing selectivity and specificity with spiked serum samples. Matrix effects were evaluated through post-column infusion experiments and comparison of standard line slopes. Precision, accuracy, and trueness were determined through an extensive 5-day protocol. Measurement uncertainty for reference value assignment was evaluated as per the Guide to the Expression of Uncertainty in Measurement (GUM), with three individual sample preparations performed on at least two different days.
Results
The RMP facilitated testosterone quantification in the range of 27.7 pmol/L (8.00 pg/mL) to 62.4 nmol/L (18.0 ng/mL) without interference from structurally-related compounds or matrix effects. Intermediate precision was ≤3.1 % and repeatability ranged from 1.4 to 1.9 % across all analyte concentrations. The bias ranged from −1.2 to 3.0 % for all levels and matrices. Expanded measurement uncertainties (k=2) for single measurements (n=1) ranged from 3.4 to 6.4 %. Measurement uncertainties for target value assignment (n=6) were ≤1.5 %, with expanded uncertainties ≤2.9 % (k=2) for all levels. Specific assessment at the LLMI yielded an expanded uncertainty (k=2) of 4.4 % for target value assignments (n=6), confirming the method’s suitability for accurate and precise quantification over the entire measuring range.
Conclusions
The RMP demonstrated high analytical performance for testosterone quantification in human serum and plasma, making it suitable for routine assay standardization and clinical sample evaluation.
Acknowledgments
We would like to thank Aline Hoffmeister, Monika Kriner, and Michael Dedio for their support in selecting and providing samples.
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Research ethics: All procedures were in accordance with the Helsinki Declaration. All samples used were exclusively anonymized samples.
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Informed consent: Not applicable.
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Author contributions: All authors have contributed to the manuscript conception and design; acquisition, or analysis and interpretation of data; drafting or revision; and final approval of the published article.
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Use of Large Language Models, AI and Machine Learning Tools: Roche Chat, Roche’s artificial intelligence (AI) Technology, was used to improve the language of the manuscript.
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Conflict of interest: Elie Fux, Rupert Schmid, Sandra Fleischer, Neeraj Singh, Christian Geletneky, Judith Taibon and Andrea Geistanger are all employees of Roche Diagnostics GmbH. Friederike Bauland is an employee of Chrestos Concept GmbH & Co. KG, Essen, Germany. Manfred Rauh and Daniel Köppl are employees of the Department of Pediatrics and Adolescent Medicine, University Hospital Erlangen. Uta Ceglarek and Alexander Gaudl are employees of the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig Medical Center. Roche employees holding Roche non-voting equity securities (Genussscheine): Elie Fux, Judith Taibon, Christian Geletneky, Andrea Geistanger, Sandra Fleischer, Rupert Schmid.
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Research funding: This research was funded by Roche Diagnostics GmbH. Manfred Rauh and Uta Ceglarek with team are funded cooperation partners of Roche Diagnostics GmbH.
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Data availability: The raw data can be obtained on request from the corresponding author.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2024-1266).
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