Reversed-phase high performance liquid chromatography as an alternative to animal bioassay for human thyrotropin potency determination

Abstract

Reversed-phase high performance liquid chromatography (RP-HPLC) was compared to the in vivo bioassay (BA) based on TSH-induced T₄ determination in mice. A linear relationship (BA_μg = 0.9790 RP-HPLC_μg − 0.052) with a highly significant correlation (r = 0.87; p < 0.001; n = 14 preparations) was found between these two methods. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for 11 other hTSH preparations was −2.01%, with a 95% confidence interval of −13.13% to +9.11%. This analysis included 5 totally or partially-degraded samples, indicating a useful correlation between the two determinations and, as expected, a higher sensitivity of the physical–chemical method. Interestingly, a commercial hTSH lot did not show any alteration 2 years post expiration date. These results demonstrate that RP-HPLC is a novel and viable alternative to the use of in vivo bioassays for hTSH potency determination, thus avoiding or reducing animal use.