Quantitative determination of trace principal components with high specific activity in menotropins
Yue Sun1, Heyang Li1, Xinyue Hu1
1National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing, China.
Introduction:
Menotropins is a mixture of gonadotropin glycoproteins extracted from the urine of postmenopausal women. Its composition is complex, with active components including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG), in addition to many unknown components. The current standards for menotropins rely exclusively on in vivo animal models for the bioactivity control of FSH and LH. However, these methods are complex and highly variable. Moreover, they fail to accurately quantify the high specific bioactivities of FSH and LH, which in turn impairs the batch-to-batch consistency of the final product. To date, there is no appropriate method for accurately quantifying these trace principal components with high specific activities.
Methods:
A reversed-phase high-performance liquid chromatography (RP-HPLC) method was established in the present study to quantify the α-subunits content common to FSH, LH, and hCG, as well as the FSH content, while simultaneously enabling purity assessment in menotropins and their formulations. The α-subunit peaks of FSH, LH, and hCG and the β-subunit peak of FSH were selected as the target peaks, with recombinant human FSH (rhFSH) employed as the external standard for quantification. Purity was calculated as the percentage of the sum of the α- and β-subunit peak areas using the area normalization method in chromatographic analysis. The validation procedure strictly followed ICH Q2(R1) guidelines and the requirements of the Chinese Pharmacopoeia, with evaluations of specificity, precision, linearity,accuracy, robustness, stability, limit of detection (LOD), and limit of quantification (LOQ). In addition to developing this HPLC method, this study conducted large-scale real-world sample analysis and head-to-head comparisons with the animal assay.
Results:
Methodological validation demonstrated that the developed RP-HPLC assay possessed excellent linearity, with correlation coefficients (R2) exceeding 0.998 for both subunits. The accuracy was confirmed by recovery rates ranging from 90.0% to 110.0%, with relative standard deviations (RSD) consistently below 2.0%. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 3 ng and 9 ng, respectively, providing the requisite sensitivity for the detection of these trace bioactive components within complex matrices. The analysis of 69 commercial batches of menotropins, sampled across manufacturing, distribution, and clinical use stages, revealed significant insights into product consistency. While standard in vivo animal bioassays exhibited substantial inter-assay variability and lacked the sensitivity to detect subtle batch-to-batch fluctuations, the HPLC method provided precise and reproducible quantification of the α-subunits (common to FSH, LH, and hCG) and the FSH.
Discussion:
As a result of its operational simplicity, accuracy, and high sensitivity, this HPLC method enables the simultaneous quantification of the total α-subunits and FSH content in menotropins, as well as assessment of their purity. It successfully addresses the analytical challenges posed by the complex matrix, abundant non-target proteins, and extremely low content of active ingredients in conventional menotropins products. It thereby provides a robust analytical tool for enhancing product quality control and safety assurance.
