Background and Aim: Determination of trace amounts of vitamins in multi-component feed premix is a troublesome analytical procedure. In this study, a simple and rapid high-performance liquid chromatography (HPLC) method was developed and validated for the concurrent detection and quantitation of four water-soluble vitamins such as thiamine, riboflavin, pyridoxine, and cyanocobalamin in veterinary feed premixes.

Materials and Methods: The chromatographic separation of the vitamins was carried out at 35°C temperature on a reversed-phase C18 column using a gradient pump mode. Mobile phase constituents were solvent (a): 25 mM Potassium dihydrogen phosphate and 5 mM sodium hexanesulfonate in deionized water having pH-4.0 and solvent and (b) 5 mM sodium hexanesulfonate in methanol. Detection was performed with HPLC ultraviolet/visible detection set at 278 and 361 nm wavelength in two different channels. The flow rate was 1.2 mL/min and the total run time was 25 min.

Results: The method was validated according to the International Conference on Harmonization and Food and Drug Administration guidelines and acceptance criteria for system suitability, precision, linearity, and recovery were met in all cases. The relative standard deviation for system suitability and precision was <2% for all vitamins. The linearity of the calibration curves was excellent (R₂>0.999) at concentration of 5, 10, 15, 20, 25, and 30 μg/mL for all vitamins. The limits of detection values were 0.0125, 0.0017, 0.0064, and 0.0065 μg/mL for thiamine, riboflavin, pyridoxine, and cyanocobalamin, respectively, and the limits of quantification values were 0.0378, 0.0051, 0.0213, and 0.0198 μg/mL for thiamine, riboflavin, pyridoxine, and cyanocobalamin, respectively. The recovery percentages ranged from 88% to 115%.

Conclusion: The overall parameters of the proposed method met the validation criteria and this method could be a highly desirable technique for routine analysis of water-soluble vitamins in veterinary feed premix.

Keywords: method development, veterinary feed-premix, water-soluble vitamin.