Validation of 5 routine assays for serum free testosterone with a candidate reference measurement procedure based on ultrafiltration and isotope dilution–gas chromatography–mass spectrometry

Background/Method: The analytical validity of free testosterone (FTe) analog immunoassays is subject to much controversy. We revisited the validation of 4 analog assays and 1 FTe calculation procedure with a metrologically traceable reference measurement procedure (RMP) based on ultrafiltration and isotope dilution–mass spectrometry for direct measurement of Te in the ultrafiltrate. To this end, we performed split-sample measurements of 40 male sera. Results: Deming regression showed that 3 of the immunoassays had moderate to good correlation (0.8474 ≤ r ≤ 0.9241) with the RMP; however, the slope was markedly below 1. The FTe calculation procedure was in good agreement with this result. The Sy/x values for all assays were higher than the combined imprecision values, which indicate their susceptibility to matrix-related effects. Conclusions: The study demonstrated substantial differences in analytical quality of FTe assays; however, the results suggested that after extending the validation with a larger variety of samples, recalibration of some analog assays might be worth further investigation.