Development and Validation of RP‐HPLC Methods for Quantifying Biotin, Vitamin D₃, and Vitamin E in Bovibest‐H Liquid

ABSTRACT

Bovibest‐H Liquid is a veterinary supplement designed to support the health and recovery of animals, particularly those with nutritional deficiencies. The goal of this study is to create and verify a reliable approach for the quantification of its active constituents, mainly biotin, vitamin D₃, and vitamin E, using reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The best chromatographic conditions were found using a Shimadzu LC‐2050 i‐series liquid chromatography system. The specifications included the use of C8 and C18 columns, isocratic elution at a flow rate of 1.0 mL/min, and detection at 254 and 200 nm. According to ICH guidelines, the methods were thoroughly validated, and the results showed that it was robust and had great recovery rates of around 99.70%, excellent specificity, linearity (R² > 0.999), precision (%RSD < 2), and valid accuracy. The present study successfully created a simple, accurate, precise, and robust HPLC method for quantifying biotin, vitamin D₃, and vitamin E. The developed methods demonstrated remarkable linearity, precision, and accuracy over a range of concentrations. This precise analytical technique could provide significant insights into future studies in the fields of veterinary sciences, pharmaceuticals, and nutritional supplements.