Alexander Bauer Westbye,a,, Oskar Kelpa,b, Yilu Yea,b, Finn Erik Aasa,b, Sandra R. Dahla,b, Per M. Thorsbya,b,c
a Hormone Laboratory, Dept. Medical Biochemistry, Oslo University Hospital
b Biochemical Endocrinology and Metabolism Research Group, Oslo University Hospital
c Institute of Clinical Medicine, University of Oslo
Email: alwest@ous-hf.no
Testosterone and the thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are key endocrine hormones regulating development, metabolism and reproductive function. Testosterone, T4 and T3 concentrations are therefore routinely assessed in clinical diagnostics. In many clinical contexts, the free (not bound to protein) hormone concentrations more accurately reflect biological activity than total levels.
Mass spectrometry (MS)-based methods following equilibrium dialysis (ED) are considered “gold standard” for free hormone quantification due to their analytical specificity and robustness against interferences. The Hormone Laboratory has recently developed a novel ED-LC-MS/MS method incorporating isotope-dilution for the simultaneous quantification of FTe, FT4, and FT3 in human serum (Fig 1). After ED, the free hormone-fraction is purified by solid phase extraction and separated on a solid-core phenyl-hexyl column. The analytes are quantitated using multiple reaction monitoring on a Sciex QTRAP 6500+, using positive electrospray ionization.
The method covered clinically relevant ranges for both sexes and was benchmarked against clinical immunoassays and estimated (calculated) FTe. To our knowledge, this is the first validated method allowing concurrent measurement of these three free hormones, supporting improved endocrine assessment in specialized clinical laboratories.
Figure 1: Free hormones (pmol/L) in human serum quantitated using LC-MS/MS A. Separation of hormones in human serum extract (free fraction). B. Quantitation of low concentrations in clinical samples.