Reverse‐Phase High‐Performance Liquid Chromatography/Mass Spectrometry (RP‐LC–MS/MS, Label‐Free Method) for Component Analysis of Gonadotropin Drugs

In assisted reproductive technology (ART), controlled ovarian hyperstimulation (COS) plays an important role in guiding treatment progress and determining success. Many ovulation-inducing agents are used in this process, including human menopausal gonadotropin (HMG), recombinant follicle-stimulating hormone (r-FSH), recombinant luteinizing hormone (r-hLH), urinary human chorionic gonadotropin (u-hCG), and recombinant human chorionic gonadotropin (r-hCG). HMG, which comes from the urine of postmenopausal women, mainly contains FSH, LH, and hCG. While bioactivity-based potency units and immunoassay-based ELISA methods remain the gold standard for quantifying protein therapeutics, LC-MS/MS is emerging as a game-changing technology. Its unique strengths are exceptional specificity and absolute quantification capabilities, independence from specialized reagents, powerful multiplexing potential, and ability to provide structural insights, establishing it as an essential tool for both orthogonal verification and cutting-edge research in the field. This study used reverse-phase liquid chromatography with mass spectrometry (RP-LC-MS/MS) and a label-free quantitative proteomics method to analyze imported r-hFSH, r-hLH, r-hCG (Gonal-F, Luveris, and Ovidrel), three domestic HMG products (Le Baode, Man Fuxin, and Xin Yunle), and u-hCG (Li Zhu). The results showed that the purity of the main component in imported gonadotropin drugs was higher than 98.55%. On the other hand, the main element of domestic u-HMG is FSH, with small amounts of hCG and very little LH. These results reveal significant differences in the composition of imported and domestic gonadotropin products, underscoring the need to further evaluate their biological activity and clinical effectiveness. This study provides important insights into drug selection for ART, with practical value for improving the quality of HMG drugs and making better use of them in clinical settings.