DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) METHOD FOR SIMULTANEOUS ESTIMATION OF NIVOLUMAB AND IPILIMUMAB IN PHARMACEUTICAL DOSAGE FORMS

Abstract

Objective: To develop and validate a simple, rapid, sensitive, precise, and accurate stability-indicating Ultra-Performance Liquid Chromatography (UPLC) method for the simultaneous estimation of Nivolumab and Ipilimumab in pharmaceutical dosage forms.

Methods: The chromatographic separation was achieved on an X bridge C18 ODS column (2.1 × 50 mm, 1.7 μm) using a mobile phase comprising phosphate buffer (pH 3.0) and acetonitrile in the ratio of 70:30 v/v at a flow rate of 0.3 mL/min. Detection was performed at 225 nm with an injection volume of 4 μL and a total run time of 4 minutes. The method was validated according to ICH Q2(R1) guidelines for specificity, linearity, accuracy, precision, sensitivity, robustness, and forced degradation studies.

Results: The optimized method demonstrated excellent system suitability with retention times of 0.685 minutes and 0.895 minutes, tailing factors of 1.14 and 1.16, plate counts of 3471 and 2143, and resolution of 4.44 for Nivolumab and Ipilimumab, respectively. The assay values were 100.25% for Nivolumab and 98.99% for Ipilimumab. Linearity was established over concentration ranges of 25–125 μg/mL for Nivolumab and 12.5–62.5 μg/mL for Ipilimumab with correlation coefficients of 0.999 for both drugs. Precision (%RSD) was 0.7% for Nivolumab and 0.1% for Ipilimumab, with intermediate precision of 0.2% for both drugs. Accuracy (mean recovery) was 99.59% for Nivolumab and 100.01% for Ipilimumab. LOD S/N ratios were 2.98 and 2.97, and LOQ S/N ratios were 9.97 and 9.98 for Nivolumab and Ipilimumab, respectively. Forced degradation studies revealed degradation ranging from 2.13% to 4.38% for Nivolumab and 7.69% to 7.87% for Ipilimumab under various stress conditions, confirming the stability-indicating nature of the method.

Conclusion: The developed UPLC method is rapid, sensitive, precise, accurate, robust, and stability-indicating for the simultaneous estimation of Nivolumab and Ipilimumab. The method is suitable for routine quality control analysis and stability studies of these immunotherapeutic agents in pharmaceutical formulations.