title: Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.
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.
authors.affiliation: Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
authors.forename: B E
authors.initials: BE
authors.lastname: Almeida
authors.forename: J E
authors.initials: JE
authors.lastname: Oliveira
authors.forename: R
authors.initials: R
authors.lastname: Damiani
authors.forename: S L
authors.initials: SL
authors.lastname: Dalmora
authors.forename: P
authors.initials: P
authors.lastname: Bartolini
authors.forename: M T C P
authors.initials: MT
authors.lastname: Ribela
mesh_terms: D006063:Chorionic Gonadotropin / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D056148:Chromatography, Reverse-Phase / Q000592:standards; D043373:Follicle Stimulating Hormone, Human / Q000032:analysis; D006801:Humans; D007986:Luteinizing Hormone / Q000032:analysis; D036361:Peptide Hormones / Q000032:analysis*; D011485:Protein Binding; D012015:Reference Standards; D012709:Serum Albumin / Q000737:chemistry*; D013972:Thyrotropin / Q000032:analysis
chemical_list: D006063:Chorionic Gonadotropin; D043373:Follicle Stimulating Hormone, Human; D036361:Peptide Hormones; D012709:Serum Albumin; D007986:Luteinizing Hormone; D013972:Thyrotropin