Measurement of Total Serum Testosterone in Adult Men: Comparison of Current Laboratory Methods Versus Liquid Chromatography-Tandem Mass Spectrometry
Abstract
The diagnosis of male hypogonadism requires the demonstration of a low serum testosterone (T) level. We examined serum T levels in pedigreed samples taken from 62 eugonadal and 60 hypogonadal males by four commonly used automated immunoassay instruments (Roche Elecsys, Bayer Centaur, Ortho Vitros ECi and DPC Immulite 2000) and two manual immunoassay methods (DPC-RIA, a coated tube commercial kit, and HUMC-RIA, a research laboratory assay) and compared results with measurements performed by liquid chromatography-tandem mass spectrometry (LC-MSMS). Deming's regression analyses comparing each of the test results with LC-MSMS showed slopes that were between 0.881 and 1.217. The interclass correlation coefficients were between 0.92 and 0.97 for all methods. Compared with the serum T concentrations measured by LC-MSMS, the DPC Immulite results were biased toward lower values (mean difference, -90 +/- 9 ng/dl) whereas the Bayer Centaur data were biased toward higher values (mean difference, +99 +/- 11 ng/dl) over a wide range of serum T levels. At low serum T concentrations (<100 ng/dl or 3.47 nmol/liter), HUMC-RIA overestimated serum T, Ortho Vitros ECi underestimated the serum T concentration, whereas the other two methods (DPC-RIA and Roche Elecsys) showed differences in both directions compared with LC-MSMS. Over 60% of the samples (with T levels within the adult male range) measured by most automated and manual methods were within +/- 20% of those reported by LC-MSMS. These immunoassays are capable of distinguishing eugonadal from hypogonadal males if adult male reference ranges have been established in each individual laboratory. The lack of precision and accuracy, together with bias of the immunoassay methods at low serum T concentrations, suggests that the current methods cannot be used to accurately measure T in females or serum from prepubertal subjects.
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Direct assays for the measurement of total and free testosterone are highly unreliable in the female range.
“Direct assays for the measurement of total and free testosterone are highly unreliable in the female range 6,7 (Grade A).”
From Global Consensus Position Statement on the Use of Testosterone Therapy for Women (2019) · cites this paper as [6]
The Discussion (p. 9) says the tested total-testosterone immunoassays lack adequate accuracy and precision for serum measurements in normal females. It says this has implications for free-testosterone determinations, but the study did not directly evaluate free-testosterone assays, so the claim about those assays is not established by this source. (LLM, conf=0.88)
Experimental — accuracy not yet measuredTotal testosterone can be measured with high accuracy and reproducibility using liquid/gas chromatography and tandem mass spectrometry assays (LC/GC-MS/MS).
“Total testosterone can be measured with high accuracy and reproducibility using liquid/gas chromatography and tandem mass spectrometry assays (LC/GC-MS/MS) 6 (Grade B).”
From Global Consensus Position Statement on the Use of Testosterone Therapy for Women (2019) · cites this paper as [6]
The LC-MSMS section (p. 3) reports accuracy, precision, and between-day reproducibility for total serum testosterone measurements, supporting the liquid-chromatography part of the claim. The paper does not establish the same performance for gas chromatography–tandem mass spectrometry; its mentions of GC-MS do not describe a GC-MS/MS assay evaluated in this study. (LLM, conf=0.87)
Experimental — accuracy not yet measuredDespite similar daily patterns, elderly males had significant blunting of peak versus trough testosterone levels, with differences of 61-205 ng/dl compared with 141-354 ng/dl in younger men.
“Despite similar patterns of variation throughout the day, elderly males experienced significant blunting of peak versus trough levels, with differences in high and low values of 61–205 ng/dl (2.1–7.1 nmol/L) compared to 141–354 ng/dl (4.9–12.3 nmol/L) in younger men.”
From Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials (2016) · cites this paper as [44]
Early studies comparing young men aged 25-27 with elderly men aged 71 found peak testosterone concentrations from 3-8 AM, extended to 2 PM in elderly men, and trough levels from early afternoon to late evening.
“Early studies comparing young (mean age 25–27 years) versus elderly males (mean age 71 years) demonstrated peak T concentrations in the 3–8 AM time period (extended to 2 PM in elderly males), with trough levels noted in the early afternoon to late evening.”
From Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials (2016) · cites this paper as [44]
Testosterone levels exhibit time-, sleep-, and age-dependent circadian variations.
“T levels exhibit time, sleep, and age-dependent circadian variations.”
From Challenges in Testosterone Measurement, Data Interpretation, and Methodological Appraisal of Interventional Trials (2016) · cites this paper as [44]
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- Authors
- Wang, C., Catlin, D. H., Demers, L. M., Starcevic, B., & Swerdloff, R. S.
- Affiliations
- University of California, Los Angeles (US)
- Pennsylvania State University (US)
- Venue
- The Journal of Clinical Endocrinology & Metabolism
- Publisher
- The Endocrine Society
- Published
- 2004-02
- Volume
- 89
- Issue
- 2
- Pages
- 534-543
- ISSN
- 0021-972X (ISSN-L)
- 1945-7197
- OpenAlex
- W2144756371
- Cited by
- 580
- References
- 25
- Citations by year
- 2026: 9 · 2025: 13 · 2024: 16 · 2023: 14 · 2022: 15 · 2021: 20 · 2020: 21 · 2019: 19 · 2018: 26 · 2017: 28 · 2016: 26 · 2015: 21 · 2014: 36 · 2013: 33 · 2012: 34
- FWCI
- 22.41
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Clinical Endocrinology & Metabolism (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Pennsylvania State University
- National Institutes of Health (5r01dk061006-04, 5r01ca071053-03)
- National Cancer Institute (R01 CA-71053)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK61006)
- National Center for Research Resources (M01 RR00543, M01RR000425, M01-RR00425)
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Health Sciences › Medicine › Reproductive Medicine › Hypothalamic control of reproductive hormones
- Keywords
- male hypogonadism, immunoassay comparison, liquid chromatography-tandem mass spectrometry, automated immunoassay, radioimmunoassay, assay bias, assay precision, reference values
- MeSH terms
- Adult, Aged, Chromatography, Liquid, Humans, Hypogonadism, Immunoassay, Male, Middle Aged, Regression Analysis, Mass Spectrometry, Testosterone, Reproducibility of Results, Case-Control Studies
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