Analytical Method Development and Validation to Determine Oxidized Follitropin Recombinant Follicle Stimulating Hormone (rFSH) Injection by RP-HPLC

Pub Date : 2024-09-05 DOI:10.2174/0115734129316027240830074632

Priyanka Chaudhary, Muthusamy Kalaivani, Pavisha Tyagi, Anubhuti Goyal, Meenakshi Dahiya, Rajeev Singh Raghuvanshi

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Abstract

Purpose: A simple, rapid and precise reverse phase–High performance liquid chromatography (RP-HPLC) method was developed for the determination of degradation impurity i.e. Oxidised follitropin in recombinant Follicle Stimulating Hormone (rFSH) Injection. Methods: Chromatographic separation was performed using a C4 column of size: 250 × 4.6 mm, 5 μm along with C3 Guard Column (SB-C3 size: 125 mm x 4.6 mm, 5 μm) and using gradient elution. The flow rate was kept at 1.0 ml per minute and the detection wavelength was at 210 nm. The retention time of oxidised follitropin was ~14 to 15 minutes. The detector showed a linear response between the range of 2.46–63.325 μg/ml (5% - 125%) with a correlation coefficient value of 0.9987. After establishing the procedure, it was ensured for its intended usage by validation of the analytical parameters like specificity, linearity, accuracy, repeatability, and robustness as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) [Q2 (R1) Validation of Analytical Procedures: Text and Methodology]. Results: All of the parameters performed using the current method yielded results that met the acceptance requirements. The detector showed a linear response between the range of 2.46– 63.325 μg/ml (5% - 125%) with a correlation coefficient value of 0.9987. Conclusions: As a result, a newly designed RP-HPLC method was capable of effectively separating impurities while maintaining acceptable limits.

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用 RP-HPLC 法测定氧化的促卵泡素重组卵泡刺激素（rFSH）注射液的分析方法开发与验证

目的：建立了一种简单、快速、精确的反相高效液相色谱（RP-HPLC）方法，用于测定重组促卵泡激素（rFSH）注射液中的降解杂质，即氧化的促卵泡激素。方法：使用 C4 色谱柱（250 × 4.6 mm，5 μm）和 C3 保护柱（SB-C3，125 mm × 4.6 mm，5 μm），采用梯度洗脱法进行色谱分离。流速保持为每分钟 1.0 毫升，检测波长为 210 纳米。氧化的促甲状腺激素的保留时间约为 14 至 15 分钟。检测器在 2.46-63.325 μg/ml（5%-125%）范围内呈线性响应，相关系数为 0.9987。在确定程序后，根据国际人用药品技术要求协调理事会（ICH）[Q2 (R1) 分析程序的验证：文本和方法]，对特异性、线性、准确性、可重复性和稳健性等分析参数进行了验证，以确保程序的预期用途。结果：使用当前方法进行的所有参数测定结果均符合验收要求。检测器在 2.46-63.325 μg/ml（5%-125%）范围内呈线性响应，相关系数为 0.9987。结论因此，新设计的 RP-HPLC 方法能够有效分离杂质，同时保持可接受的限度。

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