Testosterone (Te) and the thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are key endocrine hormones regulating development, metabolism and reproductive function. The free (not protein-bound) hormone concentrations more accurately reflect biological activity than total levels. Free hormone measurements are therefore used for clinical decision making in many contexts. Mass spectrometry (MS)-based methods following equilibrium dialysis (ED) are considered “gold standard” for free hormone quantification. Unfortunately, few clinical laboratories offer such measurements today. A 96-well format ED-LC-MS/MS method incorporating isotope-dilution for simultaneous quantitation of free Te, free T4 and free T3 in clinical samples that used offline (parallel) sample-preparation was successfully developed and validated. The method covered clinically relevant ranges, and clinical samples with a wide concentration range were analyzed to perform a comparison to existing (immunoassay or calculation) methods. Free hormone concentration in 20 healthy blood donors is reported. This is the first method to directly quantitate both free testosterone and free thyroid hormones in clinical samples. It can be implemented alongside existing LC-MS/MS methods in specialized clinical laboratories to support improved endocrine assessment and clinical decision making. • Westbye et al. • Testosterone and the thyroid hormones T4 and T3 are important endocrine hormones • Measurements of the free concentration of hormones are clinically important • Equilibrium dialysis (ED) followed by LC-MS/MS quantitation is the “gold standard” measurement method • The article reports an ED-LC-MS/MS method for simultaneous quantitation of free testosterone, free T4 and free T3 in human serum suitable for clinical chemistry • The method was extensively validated and concentrations in healthy controls are reported
Westbye et al. (Sun,) studied this question.