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Abstract: The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest. [Copyright &y& Elsevier]

Abstract: Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman–Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BAIU =0.9925 RP-HPLCIU −1.3165) with a highly significant correlation (r =0.9371; p <0.0001; n =24) was found for these two methods for six hFSH preparations of different origins. 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 six other hFSH preparations was −1.4%, with a 95% confidence interval of −9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical–chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin. [Copyright &y& Elsevier]