Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 63; pp. 160 - 164
• Main Authors: Almeida, B.E., Oliveira, J.E., Damiani, R., Dalmora, S.L., Bartolini, P., Ribela, M.T.C.P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 07-04-2012; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; bioassays; Biological and medical sciences; Chorionic Gonadotropin - analysis; Chromatography, High Pressure Liquid - standards; Chromatography, Reverse-Phase - standards; Excipient; Follicle Stimulating Hormone, Human - analysis; follicle-stimulating hormone; Fundamental and applied biological sciences. Psychology; General pharmacology; Glycoprotein hormones; glycoproteins; HSA; human chorionic gonadotropin; human serum albumin; Humans; ingredients; luteinizing hormone; Luteinizing Hormone - analysis; Medical sciences; Peptide Hormones - analysis; Pharmacology. Drug treatments; Protein Binding; quantitative analysis; Reference Standards; reversed-phase high performance liquid chromatography; RP-HPLC; Serum Albumin - chemistry; thyrotropin; Thyrotropin - analysis; WHO standards; RP-HPLC; WHO standards; Excipient; Glycoprotein hormones; HSA; Human; Reversed phase chromatography; Vehicle(excipient); HPLC chromatography; Serum albumin; Glycoprotein hormone; Quantitative analysis
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2012.01.039

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.