Reassessing free testosterone calculation by liquid chromatography-tandem-mass-spectrometry direct equilibrium dialysis.

BACKGROUND: Assessment of free testosterone (FT) can help to evaluate androgen status in subjects with borderline total testosterone (T) and/or altered SHBG levels. However, there has been ongoing debate as to the validity of different methods to measure or calculate FT, resulting in uncertainty. METHODS: 183 women and 146 men were analysed using equilibrium dialysis with direct FT measurement by LC/MS-MS. FT calculation was re-evaluated for the mass action lawbased equation according to Vermeulen (FTC-V), empirical equations according to Ly (FTC-L) and a recently proposed calculation based on a multi-step, dynamic, allosteric model according to Zakharov (FTC-Z). RESULTS: Median FT in females was 13 pmol/L [1.2% of T], in males 248 pmol/L [1.5% of T]. In women absolute FT and %FT were strongly inversely correlated to SHBG and significantly to albumin. %FT was unrelated to T. In men %FT was strongly inversely correlated to both SHBG and T. FT was weakly associated with SHBG and largely independent of T and albumin. Rank correlations for FTC% versus equilibrium dialysis FT% (data for men) are 0.62, 0.74 and 0.89 for FTC-Z, FTC-L and FTC-V. Between calculations there are differences in accuracy and in dependency of their performance on SHBG and T levels. CONCLUSION: Direct equilibrium dialysis results for FT support previous evidence from indirect equilibrium dialysis and ultrafiltration methodologies. FTC as surrogate of measured FT has inherent limitations with clinically important differences among evaluated equations.r. FTC-V showed the most robust approximation of FT across a broad range.