Using LC-MS/MS to Measure Testosterone in Dried Blood Spots

Testosterone levels have traditionally been measured using serum or plasma samples—which presents challenges for sample collection, storage, and transport, particularly in resource-limited settings. However, dried blood spot (DBS) sampling has emerged as an effective alternative for hormone analysis, offering significant advantages for sample stability, ease of collection, and simplified logistics.

A recent study conducted by Yehudah Gruenstein of the Department of Biochemistry and Molecular Biology, at the University of Miami (Coral Gables, Florida) aimed to validate a DBS-based testosterone assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to ensure accuracy and precision comparable to conventional serum-based methods. A paper based on this study was published in Analytical Science Advances (1).

As a key androgen hormone, testosterone plays a major role in the regulation of numerous physiological functions in both men and women, including, but not limited to, reproductive health, muscle mass, bone density, and overall well-being (2,3). The accurate and reliable measurement of testosterone levels is crucial in clinical settings, especially for the diagnosis of such conditions as hypogonadism, polycystic ovary syndrome, and other endocrine disorders (2,4). Traditionally, testosterone measurements are performed using serum or plasma, but this present several logistical challenges, especially for sample collection, storage, and transport, which become more pronounced in resource-limited settings or in large-scale epidemiological studies.

To address these limitations, alternative methods such as DBS sampling have gained significant traction in recent years for hormone analysis, including testosterone (5–7). While recent advancements in analytical techniques, particularly those using liquid chromatography-tandem mass spectrometry (LC-MS/MS), have dramatically heightened the sensitivity and specificity of testosterone assays, including those using DBS (5,8), Gruenstein believed that a standardized approach to DBS assay validation for testosterone measurement for routine testing remained necessary.

Gruenstein’s findings from the validation of the testosterone assay demonstrate that the method is robust, accurate, and precise across a wide range of conditions. The assay showed excellent linearity with a correlation coefficient (r²) of 0.999 across testosterone concentrations ranging from 0.1 to 100 ng/mL. The evaluation of inter- and intra-day precision further supports the robustness of the assay. Inter-day precision, assessed over 5 days at low, medium, and high testosterone concentrations, demonstrated %CV values below 10% for all concentrations. Similarly, the intra-day precision evaluation showed %CV values below 5%, further indicating the reliability of the method in providing consistent results within a single day. The lower limit of quantification and the limit of detection were calculated at 0.086 and 0.058 ng/mL, respectively, confirming the assay's sensitivity in detecting low testosterone concentrations. These results are aligned with established validation guidelines, ensuring the method's suitability for clinical and research applications where accurate testosterone quantification at low concentrations is required. Matrix effect and recovery assessments confirmed that the method is resistant to matrix effects, with acceptable recoveries of 75% at low concentrations and 81% at high concentrations (1).

Gruenstein said that this testosterone assay exhibits high accuracy, precision, and stability, with minimal matrix effects or interference, and is suitable for use in clinical and research applications where reliable routine testing of testosterone quantification is essential (1).

Testosterone Molecule Art. © ArtiStock - stock.adobe.com

References

1. Gruenstein Y. Validation of a Dried Blood Spot Assay for Testosterone Measurement Using Liquid Chromatography-Tandem Mass Spectrometry. Anal. Sci. Adv. 2024, 5 (11–12), e202400035. DOI: 10.1002/ansa.202400035

2. Kanakis, G. A.; Tsametis, C. P.; Goulis, D. G. Measuring Testosterone in Women and Men. Maturitas 2019, 125, 41–44. DOI: 10.1016/j.maturitas.2019.04.203

3. Nieschlag, E.; Behre, H. M.; Nieschlag, S. (Eds.). Testosterone: Action, Deficiency, Substitution. Cambridge University Press, 2012.

4. Hahn, S.; Kuehnel, W.; Tan, S.; Kramer, K.; Schmidt, M.; Roesler, S.; Kimmig, R.; Mann, K.; Janssen, O. E. Diagnostic Value of Calculated Testosterone Indices in the Assessment of Polycystic Ovary Syndrome. Clin. Chem. Lab. Med. 2007, 45 (2), 202–207. DOI: 10.1515/CCLM.2007.031

5. Desai, R.; Savkovic, S.; Handelsman, D. J. Dried Blood Spot Sampling of Testosterone Microdosing in Healthy Females. J. Steroid Biochem. Mol. Biol. 2024, 240, 106496. DOI: 10.1016/j.jsbmb.2024.106496

6. Marshall, D. J.; Adaway, J. E.; Hawley, J. M.; Keevil, B. G. Quantification of Testosterone, Androstenedione and 17-Hydroxyprogesterone in Whole Blood Collected Using Mitra Microsampling Devices. Ann. Clin. Biochem. 2020, 57 (5), 351–359. DOI: 10.1177/0004563220937735

7. Grecsó, N.; Zádori, A.; Szécsi, I.; Baráth, Á.; Galla, Z.; Bereczki, C.; Monostori, P. Storage Stability of Five Steroids and in Dried Blood Spots for Newborn Screening and Retrospective Diagnosis of Congenital Adrenal Hyperplasia. PLoS One 2020, 15 (5), e0233724. DOI: 10.1371/journal.pone.0233724

8. Skogvold, H. B.; Rootwelt, H.; Reubsaet, L.; Elgstøen, K. B. P.; Wilson, S. R. Dried Blood Spot Analysis with Liquid Chromatography and Mass Spectrometry: Trends in Clinical Chemistry. J. Sep. Sci. 2023, 46 (15), 2300210. DOI: 10.1002/jssc.202300210