DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ATAZANAVIR SULPHATE AND RITONAVIR IN BULK AND TABLET DOSAGE FORMS

A robust and validated reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of Atazanavir Sulphate and Ritonavir in bulk and tablet dosage forms. The development process involved a systematic optimization of chromatographic parameters, including the selection of wavelength, mobile phase composition, flow rate, and column. UV-spectrophotometric analysis revealed maximum absorbance for Atazanavir Sulphate and Ritonavir at 249 nm and 239 nm, respectively. However, a detection wavelength of 210 nm was chosen for HPLC due to optimal peak response for both drugs. Several chromatographic trials were conducted to optimize the conditions. The final optimized method employed a Nucleodur C18 column (150 mm × 4.6 mm × 5 µm), with a mobile phase composed of acetonitrile, methanol, and phosphate buffer (pH 3.0 adjusted with orthophosphoric acid) in the ratio 44:11:45 v/v/v. The flow rate was maintained at 1.5 mL/min and the injection volume was 20 µL. Under these conditions, Atazanavir and Ritonavir were eluted with sharp, symmetrical peaks and good resolution, with retention times of approximately 3.13 and 6.10 minutes, respectively. Method validation was performed in accordance with ICH guidelines. Specificity studies confirmed the absence of interference from excipients, with no peaks observed in blank or placebo chromatograms. System suitability parameters, including theoretical plates, resolution, tailing factor, and symmetry factor, were all within acceptable limits, confirming the adequacy of the system. Linearity was demonstrated for Atazanavir Sulphate (34–102 µg/mL) and Ritonavir (10–30 µg/mL), with correlation coefficients (r²) of 0.999 for both drugs. Precision was established by evaluating method repeatability; %RSD values were 0.12% for Atazanavir and 1.39% for Ritonavir, indicating excellent reproducibility. Accuracy was confirmed through recovery studies at 80%, 100%, and 120% levels, yielding recoveries within the range of 99.91% to 100.40% for Atazanavir Sulphate and 100.12% to 100.31% for Ritonavir. Robustness was evaluated by altering flow rates slightly (±0.1 mL/min), and the method showed negligible variations with %RSD well below 2%, proving its consistency. Ruggedness, assessed by intraday and intermediate precision with different analysts, yielded %RSD values below 1% for both drugs, confirming the method's analyst-independence and reproducibility. The method also demonstrated high sensitivity with limits of detection (LOD) and quantitation (LOQ) of 0.002 µg/mL and 0.016 µg/mL for Atazanavir Sulphate, and 0.001 µg/mL and 0.004 µg/mL for Ritonavir, respectively. In conclusion, the developed RP-HPLC method is simple, accurate, precise, rapid, and cost-effective. Its high sensitivity, robustness, and reproducibility make it highly suitable for routine quality control analysis of Atazanavir Sulphate and Ritonavir in both bulk drug substances and tablet dosage forms.