Development and Validation of Analytical Methods by RP-HPLC for Estimation of Vitamin D3 and Biotin in Calpond Gold Gel Suspension and its Stability at Accelerated Conditions

Introduction: In the current research, simple and isocratic analytical methods were developed and validated by ICH Q2 (R1) guidelines for the accurate quantitative measurement of Vitamin D3 and Biotin in Calpond Gold Gel suspension. Methods: The analytical methods were developed using RP-HPLC with the best chromatographic conditions. These methods were validated for system suitability, specificity, linearity, LOD, LOQ, accuracy, precision, and robustness. Moreover, an accelerated stability study was carried out for Vitamin D3 and Biotin to estimate the rate of degradation at accelerated conditions (Temp. 40°C ± 2°C and RH 75% ± 5% for 6 Months). Results: Methods showed good suitability (tailing factor ˂ 2%, Theoretical plates ˃ 2000), robustness and specificity without any significant interference, exhibited good linearity (R2 ˃ 0.990) over concentration ranges (3200 – 9600) IU for Vitamin D3 and (12.5 – 37.5) μg/ml for Biotin, precision (RSD ˂ 2%), recovery rates (˃ 99%), and LOD, LOQ were found to be 608.79 IU, 1844.82 IU and 1.72 μg/ml, 5.22 μg/ml for Vitamin D3 and Biotin. Additionally, the % Assay complied with the in-house acceptance criteria (˂ 90%) of label claim from 0-6 months. Discussion: The developed methods were found in accordance with ICH Q2 (R1) guidelines. The validation parameters remained within the acceptable limits, indicating that the methods are reliable for the quantitative determination of Vitamin D3 and Biotin in Calpond Gold Gel suspension. Moreover, Vitamin D3 and Biotin were found to be stable during the accelerated stability study. Conclusion:: The simple and isocratic RP-HPLC methods were successfully developed and validated according to the current ICH guidelines. The developed methods might be useful for the estimation of vitamin D3 and Biotin in feed supplements, veterinary, and pharmaceutical formulations.