Abstract
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.
Highlights
Vitamins are critical and biologically active organic compounds that are minor but essential constituents of food required for normal metabolism in human and animal bodies [1,2]
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 system suitability parameters (Table-1) reveal that the percentage of relative standard deviation (RSD) for retention time and peak area (PA) is 2, which prove that the values are within the specified limits of the validation process [21,22,23]
Summary
Vitamins are critical and biologically active organic compounds that are minor but essential constituents of food required for normal metabolism in human and animal bodies [1,2]. Even the premixes are designed for the market or used directly for feed production; it is necessary to achieve homogeneity and well balanced of all ingredients. Different types and quantities of micro-ingredients such as vitamins have been used in feed premix production depending on species and categories of animals [4]. Contemporary feed production is inconceivable without adding vitamin-mineral premixes. A trace amount of vitamins is required. Determination of trace amounts of vitamins in multi-component feed premix is a troublesome analytical procedure. 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
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