Development and Validation of a Robust RP-HPLC Method for Simultaneous Analysis of Thiamine, Niacinamide, and Pyridoxine in Mecovet-XL Syrup and its Stability at Accelerated Conditions

Introduction:: The present study aimed to develop a simple, reliable, and robust analytical method for the simultaneous quantification of Vitamins B1, B3, and B6 in Mecovet-XL syrup. The developed method was thoroughly validated by ICH Q2 guidelines to ensure its suitability for accurate and consistent quantitative analysis. Methods:: An RP-HPLC method was optimized under suitable chromatographic conditions. The method validation included evaluation of system suitability, specificity, linearity, LOD, LOQ, accuracy, precision, and robustness. Furthermore, an accelerated stability study was performed to assess the stability of Vitamins B1, B3, and B6 under elevated temperature and humidity conditions. Results:: The method demonstrated excellent suitability, robustness, and specificity without any notable interference, and exhibited good linearity (R2 ˃ 0.990), precision (RSD ˂ 2%), and recovery rates (˃ 98%). LOD and LOQ were found to be 2.33 μg/ml, 3.65 μg/ml, 0.81 μg/ml, 7.07 μg/ml, 11.08 μg/ml, and 0.24 μg/ml for Vitamins B1, B3, and B6. Additionally, the % assay results indicated a slower degradation rate during accelerated stability conditions. Discussion:: The developed method was found to be in accordance with the ICH Q2 guidelines. The validation parameters remained within acceptable limits, indicating that the method is reliable for the simultaneous quantification of Vitamins B1, B3, and B6. Moreover, these vitamins were found to be stable during the accelerated stability study. Conclusion:: The developed RP-HPLC method proved to be reliable, precise, robust, and can be effectively applied in future research for the quantification and stability assessment of these vitamins in feed supplements, veterinary, and pharmaceutical formulations containing complex multivitamin compositions.