Loureiro Renan Fernandes, de Oliveira João Ezequiel, Torjesen Peter A, Bartolini Paolo, Ribela Maria Teresa C P
Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
J Chromatogr A. 2006 Dec 8;1136(1):10-8. doi: 10.1016/j.chroma.2006.09.037. Epub 2006 Oct 17.
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.
建立了一种反相高效液相色谱（RP-HPLC）法用于完整人促卵泡激素（hFSH）的定性和定量分析，并对其准确性、精密度和灵敏度进行了验证。人促卵泡激素是一种二聚体糖蛋白激素，在生殖医学中广泛用作诊断分析物和治疗产品。所开发的技术保留了蛋白质的完整性，能够分析完整的异二聚体形式，而不像目前大多数使用的条件那样仅分析其亚基。该方法还用于比较垂体、尿液和两种中国仓鼠卵巢（CHO）来源的hFSH制剂以及垂体前叶的另外两种相关糖蛋白激素：人促甲状腺激素（hTSH）和人促黄体生成素（hLH）的相对疏水性。所分析的三种糖激素中疏水性最小的是hFSH，其次是hTSH和hLH。在垂体和重组hFSH制剂之间观察到t(R)有显著差异（p<0.005），反映了它们碳水化合物部分的结构差异。在尿液hFSH中检测到两种主要的异构体，其中一种与垂体和重组制剂有显著差异（p<0.005）。证明了该RP-HPLC方法的剂量-反应曲线的线性（r=0.9965，n=15），以及不同hFSH制剂定量的批间精密度小于4%和灵敏度约为40 ng。还分析了垂体和重组制剂单个亚基的色谱行为和相对疏水性。此外，通过基质辅助激光解吸电离飞行时间质谱分析（MALDI-TOF-MS）同时测定了单个hFSH亚基和异二聚体的分子量。我们认为，本方法是该激素表征和质量控制的重要工具，主要药典中尚未对此进行描述。
