Simultaneous quantitation of free testosterone and free thyroid hormones in human serum by equilibrium dialysis...

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 method allows direct quantitation of both free testosterone and free thyroid hormones in human samples at clinically relevant concentrations. It can be implemented alongside existing LC-MS/MS methods in specialized clinical laboratories to support improved endocrine assessment and clinical decision making.