Quantitative Analysis of Fat-Soluble Vitamins in Feed Additives Using an In-House Developed and Validated HPLC Method

Abstract

Quantification of vitamins in complex matrices such as feed additives is a time-consuming analytical procedure. In this study, a simple and precise in-house High Performance Liquid Chromatography (HPLC) method was developed and validated for the simultaneous detection and quantification of four fat-soluble vitamins such as vitamin A, D3 , E, and K3 in feed additives. The HPLC method was developed and validated using reversed-phase column chromatography. The chromatographic separation of the vitamins was carried out at 25° C temperature on a reverse-phase C18 column using a binary gradient pump mode. Mobile phase constituents were solvent (a): deionized water and (b) methanol. Detection was performed with HPLC ultraviolet/visible detection set at 325, 265, 230, and 254 nm wavelength for vitamin A, D3 , E, and K3 respectively. The flow rate was 1.0mL/min and the total run time was 20min. The method was validated according to the guidelines of the International Conference on Harmonization (ICH) and Food and Drug Administration (FDA), USA, and acceptance criteria for system suitability, specificity, linearity, accuracy, and precision were met in all the cases. The Relative Standard Deviation (RSD) for system suitability and precision was <2% for all the studied vitamins. The linearity of the calibration curves was excellent (R2 >0.999) at concentrations of 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 µg/mL for all vitamins, and the range of linearity of this method was 0.0-50.0 μg/mL with R2 value greater than 0.999. The limits of detection values were 0.0022, 0.0012, 0.0022, and 0.0020 µg/ mL for vitamin A, D3 , E, and K3 , respectively, and the limits of quantification values were 0.0066, 0.0038, 0.0066, and 0.0061 µg/mL for vitamin- A, D3 , E, and K3 respectively. The recovery percentages ranged from 85% to 103%, and the robustness of the method is also high with excellent reproducibility. The overall parameters of the proposed method met the validation criteria and this method could be a precise and highly desirable analytical procedure for accurate quantification of four fat-soluble vitamins such as A, D3 , E, and K3 in feed additives using a single chromatographic run.