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Requirement for Mass Spectrometry Sex Steroid Assays in the Journal of Clinical Endocrinology and Metabolism
Abstract
CLICK HERE FOR AN UPDATE ON THE REQUIREMENT FOR MASS SPECTROMETRY SEX STEROIDS ASSAYS The accurate measurement of hormones is the pivot of modern endocrinology. Although tracing its roots to ancient writings, the modern scientific discipline came into existence after a series of mid-20th century Nobel Prize-winning achievements in identifying and measuring hormones. Arguably the most decisive step was the invention of immunoassay in 1959 (1), originally for peptide hormones but extended a decade later to nonimmunogenic steroids (2). This armed the nascent discipline with revolutionary impact through the ability to measure virtually every hormone at orders of magnitude lower than had ever been possible before with earlier cumbersome, insensitive, whole-animal bioassays. Yet the pioneers of steroid immunoassay understood its limitations as that decade's delay was consumed in finding the additional workarounds required to create valid formats for immunoassay of small, subimmunogenic molecules like steroids. These essentials comprised solvent extraction, chromatography, and structurally authentic tracers, a triplet of validity criteria. Employing these required skills originally confined steroid measurement to manual methods in specialized laboratories; however, in the 1980–1990s, the steeply increasing demand for steroid measurement in the clinic and laboratory drove assay simplification to adapt steroid immunoassays into popular one-step kits and multiplex assay platform formats. This commodification resulted in “direct” steroid immunoassays, which bypassed all the original triplet of validity criteria (most fundamentally extraction), sacrificing accuracy and specificity for throughput speed and lower cost. Although problems with validity (analytical specificity, accuracy) of direct immunoassays were soon identified and were reported over 25 years ago (3, 4), the fundamental significance of violating these validity criteria resurfaced prominently in the last decade as the accuracy of direct hormone assays came under more stringent scrutiny—and as a solution, affordable and accessible mass spectrometry (MS) steroid assays came into view. The method-specific bias and analytical nonspecificity of direct T immunoassays are worst at the low circulating T levels in children, women, and men with pathological, functional, or experimentally induced T deficiency (5–7) where such direct immunoassays have been famously likened to random number generation (8). By modern standards, putative findings using such suboptimal methods would require verification by more specific MS-based assays. This renders direct T immunoassays methodologically inadequate for high-quality clinical reproductive medicine research. Not surprisingly, aiming to measure circulating (picomolar) estradiol concentrations 10- to 100-fold lower than circulating (nanomolar) T levels that render direct T immunoassays unreliable is clearly problematic. Thus, direct estradiol immunoassays also lack accuracy in children (9), men (10, 11), and postmenopausal (3, 12–15) or aromatase inhibitor-treated (16) women who all share comparably low circulating estradiol concentrations. It is beyond doubt now that direct immunoassays of sex steroids are methodologically inadequate for high-quality clinical research. MS-based steroid measurements have been available for decades, indeed as long as immunoassays (17). Although they always remained the reference method for steroid specificity and structure, MS-based steroid assays were always too expensive and unavailable for routine use in the laboratory to support clinical research because they required highly skilled operators using complex, difficult-to-access equipment. Hence, while remaining the reference method for steroids, MS was too insensitive, expensive, and unavailable for routine clinical research use. All of this changed over the last decade with the advent of bench-top mass spectrometers with sensitivity matching the best immunoassays while retaining reference level specificity and introducing multianalyte capability. Not only does the availability of MS-based steroid assays resolve the problems of direct steroid immunoassays, but multianalyte profiling also ushers in a new era of snapshot profiling of the overlooked importance of steroid pathway fluxes that will broaden the interpretation of clinical studies beyond the standard restricted single analyte-by-analyte analysis. In this context, it is salient that the nonspecificity and single analyte limitations of steroid immunoassays always rendered them inadequate for antidoping testing where MS-based profiling has been standard for decades. Where an elite athlete's pursuit of his or her profession could be ended by a positive antidoping test, detection of banned steroids had to be undertaken at the highest standards of scientific rigor to withstand the most vigorous medico-legal challenge. It is time that clinical research and practice operates under equally h
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“However, caution is needed when interpreting these data as radioimmunoassays (n = 10) and chemiluminescence immunoassays (n = 9) were predominately used for the measurement of androgens, which are less sensitive than mass spectrometry (n = 2) in detecting androgen levels in women (Handelsman and Wartofsky, 2013).”
From Insulin resistance in polycystic ovary syndrome: a systematic review and meta-analysis of euglycaemic–hyperinsulinaemic clamp studies (2016) · cites this paper as Handelsman and Wartofsky, 2013
In the citing paper
“Our results regarding varying serum TT levels assessed via immunoassay and ID-LC–MS/MS supports previous statements on the unreliable results of the currently used immunoassays [25] as well as the need for at least two measurements of low TT levels to confirm the diagnosis of hypogonadism [26].”
From Effects of vitamin D supplementation on androgens in men with low testosterone levels: a randomized controlled trial (2019) · cites this paper as [25]
In the citing paper
“The Endocrine Society has now established minimum performance criteria for steroid assays, which recognize that metrological traceability is not currently available for all assays, yet remains an important goal (12)(13)(14).”
From Serum cortisol: an up-to-date assessment of routine assay performance (2016) · cites this paper as [12]
In the citing paper
“A reliable threshold for circulating testosterone must be set using measurement by the reference method of liquid chromatography–mass spectrometry (LC-MS) rather than using one of the various available commercial testosterone immunoassays.”
From Circulating Testosterone as the Hormonal Basis of Sex Differences in Athletic Performance (2018) · cites this paper as [23]
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“Recognizing the limitations of direct steroid immunoassays, The Endocrine Society commissioned two Position Papers written by experienced immunoassayists on the challenges to T (18) and estradiol (19) measurement and supported a renewed effort to standardize T assays (20).”
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Metadata
- Authors
- Handelsman, D. J., & Wartofsky, L.
- Affiliations
- The University of Sydney (AU)
- MedStar Washington Hospital Center (US)
- Corresponding author
- Handelsman, D. J.
- Venue
- The Journal of Clinical Endocrinology & Metabolism
- Publisher
- The Endocrine Society
- Published
- 2013-10-01
- Volume
- 98
- Issue
- 10
- Pages
- 3971-3973
- ISSN
- 0021-972X (ISSN-L)
- 1945-7197
- OpenAlex
- W2116024161
- Cited by
- 368
- References
- 34
- Citations by year
- 2026: 21 · 2025: 21 · 2024: 24 · 2023: 25 · 2022: 23 · 2021: 26 · 2020: 30 · 2019: 22 · 2018: 35 · 2017: 35 · 2016: 33 · 2015: 43 · 2014: 26 · 2013: 2
- FWCI
- 24.07
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Clinical Endocrinology & Metabolism (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Health Professions › Medical Laboratory Technology › Clinical Laboratory Practices and Quality Control
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
- Keywords
- testosterone measurement, estradiol measurement, assay standardization
- MeSH terms
- Humans, Immunoassay, Gonadal Steroid Hormones, Mass Spectrometry, Editorial Policies
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