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.

Keywords: Recombinant follicle stimulating hormone (rFSH), oxidized impurities, follitropin injection, validation and verification, RP-HPLC, ICH Q2(R1).