Development and Validation of a Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Trastuzumab and Hyaluronidase-OYSK in Pharmaceutical Dosage Forms

Research Article

Abstract

The precise quantification of biologic combinations in pharmaceutical formulations necessitates robust and sensitive analytical techniques that can withstand the complexity of macromolecular structures. A simple, rapid, and reproducible Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Trastuzumab and Hyaluronidase-OYSK. Chromatographic separation was successfully achieved utilizing a Waters Alliance-e2695 system equipped with a Luna Phenyl-Hexyl column (250 x 4.6 mm, 5 µm). The isocratic mobile phase consisted of Acetonitrile and 0.1% Trifluoroacetic acid (TFA) in an equal ratio of 50:50 %v/v, pumped at a flow rate of 1.0 mL/min. Detection was monitored at 228 nm using a photodiode array detector at ambient temperature. The developed method demonstrated superior system suitability, with the number of theoretical plates exceeding 2000 and a tailing factor maintained below 2.0 for both analytes. Linearity was established across significant concentration ranges, and the correlation coefficient was found to be greater than 0.999. Precision was confirmed with a Relative Standard Deviation (RSD) of peak areas consistently below 2.0%. The method was extensively validated in accordance with International Council for Harmonisation (ICH) guidelines, covering parameters such as accuracy, precision, specificity, robustness, and sensitivity. Moreover, forced degradation studies under various stress conditions acidic, alkaline, thermal, oxidative, and photolytic revealed that the method effectively separates degradation products from the main analytes. These results indicate that the proposed RP-HPLC method is suitable, economical, and precise for the routine quality control and stability assessment of Trastuzumab and Hyaluronidase-OYSK in pharmaceutical dosage forms.