SIMULTANEOUS ANALYSIS METHOD OF TEN WATER-SOLUBLE VITAMINS WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN FISH FEED ADDITIVES

Abstract

Water-soluble vitamins (WSVs) as feed additives are useful for increasing growth and immunity against diseases. Analysis of WSVs has been carried out using HPLC with fluorescence or diode arrays detector. Currently, there is no simultaneous analysis method for 10 WSVs with an HPLC UV detector at two wavelengths detection. This study aimed to develop a simultaneous analysis method for 10 WSVs (vitamin B1, B2, B5, B6, B7, B9, B12, ascorbic acid, niacin, and nicotinamide) with an HPLC-UV at 210 and 254 nm. The results showed that this method was able to analyze 10 WSVs in 25 min, with an isocratic mobile phase (24:76) consisting of methanol (A) and 0.05 M phosphate buffer pH 3.0 containing 0.01 M sodium 1-hexane sulfonate and 0.1% TEA (B), Shimpack C18 (250 mm x 4.6 mm, 5μm) column, at flow rate 1 ml/min. The method has been validated with parameters based on the Association of Official Analytical Chemists (AOAC). The method has good specificity with resolution >1.15. Linearity was expressed as a correlation coefficient of r > 0.99. The LOD ranged from 0.71 mg/L and 16.32 mg/L; whereas the LOQ ranged from 2.23 mg/L and 54.40 mg/L. Intra-day and inter-day precision were expressed as relative standard deviations in the range of 0.16% to 4.18% and 0.40% to 5.59%, respectively. While the accuracies were in the range of 81.6% to 109.71%. The method can be used to quantify 10 WSVs in fish feed additives for routine analysis in the laboratory.