EndoAxis Clinical Team
At EndoAxis, we want to utilize appropriate data and analysis for values that have been well-researched and provide clinical insight. But methodology matters when assessing hormones.
Why Free Testosterone (FT) Matters
Free testosterone (FT) is one such value that warrants appropriate assessment.
FT measurements can help clarify cases of hyperandrogenism in women and hypogonadism in men—especially when total testosterone levels are borderline or SHBG is altered. The free testosterone fraction is a very small portion of testosterone circulating, so precision and excretion are critical in identifying appropriate levels.
The gold-standard methods used in the research field are equilibrium dialysis (ED) and ultrafiltration (UF). However, these methods are technically demanding, labor intensive, expensive, and not well suited for high-volume clinical use.
The Problem With Commercial Lab Tests
Many commercial labs testing free testosterone offer direct-immunoassay (analog) FT tests. These methods, while convenient, fast and cheap, suffer from significant inaccuracy and potential cross-reactivity. They are widely regarded as unreliable, particularly at the low concentrations found in women, children, or androgen-deficient individuals. Consequently, researchers and guideline bodies strongly caution against their clinical use (1, 2, 3).
The Rise of LC-MS/MS & Its Clinical Promise
Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has increasingly been recognized as a more specific, accurate, and sensitive method for measuring both total and free testosterone outside of the research labs—especially at lower concentrations. Clinical guidelines support the use of LC-MS/MS confirmation for testosterone values if total testosterone is inconsistent with the clinical picture (3).
Why the Calculated Approach Still Holds Its Ground
Multiple studies affirm that calculated FT, when performed with validated formulas such as the Vermeulen method, aligns closely with FT values obtained via LC-MS/MS or equilibrium dialysis (4).
Interestingly, the Vermeulen calculation is so consistent with common LC-MS/MS free testosterone results that, for screening purposes, many clinicians prefer the calculated result over a lab’s direct FT measurement. This makes calculated FT not only a practical alternative, but in many cases, the more trustworthy front-line tool.
The Endocrine Society, the European Academy of Andrology, and the European Society of Endocrinology all recommend calculated FT (derived from TT, SHBG, and albumin) over direct analog assays, particularly in borderline cases or when SHBG is abnormal. This reflects a broad consensus that methodology matters more than convenience when measuring such a small hormone fraction (1, 5, 6).
In the EndoAxis platform, users are able to provide a total testosterone and sex hormone binding globulin (SHBG) level for comparison to the urinary bioavailable testosterone reported on the DUTCH test. As not all providers have a recent metabolic panel to add in albumin, but may still have a total testosterone and SHBG level, we have opted in our analysis to use an average albumin. For ease of data entry and consistency across report analyses, we apply a standardized albumin value of 4.3 g/dL—within the normal physiological range of 3.4–5.4 g/dL. The Vermeulen equation accounts for a testosterone binding affinity quotient used for both SHBG and albumin; however, because testosterone binds only weakly to albumin, variations in albumin within the normal range have a negligible effect on calculated free testosterone (≈0.1 ng/dL). Using a fixed albumin average provides a consistent and clinically valid comparison to urinary bioavailable testosterone, even when recent serum albumin levels are unavailable. As we expand our analysis, the albumin-specific values will be incorporated for greater precision.
EndoAxis Approach: Balanced, Method-Driven, Clinically Prudential
Your EndoAxis system embodies a forward-thinking approach.
Calculated FT remains central, given its proven alignment with both LC-MS/MS and research-grade methods. Methodology awareness is key—clinicians must recognize that FT is a tiny fraction of total testosterone, requiring precision in measurement. Immunoassay will not provide accurate information.
LC-MS/MS integration ensures alignment with gold-standard research techniques. But this method is not the default at most lab companies, meaning you must—as the provider—select the appropriate lab from their catalogue.
An additional caveat is that if you are running the lab through insurance, some insurance companies will deny LC-MS/MS due to cost.
For now, calculated FT will continue to be our primary clinical marker, with LC-MS/MS used as a confirmatory or high-precision tool.
