Analysis of intact human follicle-stimulating hormone preparations by reversed-phase high-performance liquid chromatography.
A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as a therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in t(R) between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) from the pituitary and recombinant preparations. The linearity of the dose-response curve (r=0.9965, n=15) for this RP-HPLC methodology, as well as an inter-assay precision of less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behaviour and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the molecular mass of individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for the characterization and quality control of this hormone, that is not yet described in the main pharmacopoeias.
Loureiro
Renan Fernandes
RF
Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
de Oliveira
João Ezequiel
JE
Torjesen
Peter A
PA
Bartolini
Paolo
P
Ribela
Maria Teresa C P
MT
D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005640:Follicle Stimulating Hormone / Q000032:analysis*; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005640:Follicle Stimulating Hormone