Background: Trastuzumab and hyaluronidase-oysk combination represents an innovative subcutaneous cancer therapy requiring robust analytical methods for quality control. Objective: To develop and validate a reversed-phase high-performance liquid chromatography method for simultaneous quantification of trastuzumab and hyaluronidase-oysk. Methods: Systematic optimization using Waters XBridge BEH300 C8 column with gradient elution of 0.1% trifluoroacetic acid in water and acetonitrile. Detection at 280 nm, flow rate 1.0 mL/min, column temperature 35°C. Validation performed according to ICH Q2(R1) guidelines. Results: Excellent separation achieved with resolution >5.0. Linearity demonstrated with correlation coefficients >0.999 for both analytes (trastuzumab: 0.5-1.5 mg/mL; hyaluronidase-oysk: 0.1-0.3 mg/mL). Mean recoveries were 99.91% and 99.92% respectively. Precision studies showed RSD <0.2%. Method proved stability-indicating through forced degradation studies. Pharmaceutical dosage form analysis yielded results within 0.1% of label claims.

Trastuzumab, Hyaluronidase-oysk, RP-HPLC, Method validation, Simultaneous estimation