Development of Method to Simultaneous Determination of Some Water and Fat Soluble Vitamins in Feeding Additives

Abstract

This study was designed in order to develop a new method to simultaneously determine the level of water and fat soluble vitamins in feed additives. In this study, we tried to measure the levels of vitamins C, B₂, B₆, and B₁₂ and nicotinic acid and folic acid (i.e., water soluble vitamins) and vitamins A, D₃, K₃, and E (i.e., fat soluble vitamins). Several extraction procedures were performed in order to extract vitamins from tablets and the most appropriate solution for this purpose was determined as 0.01% aqueous trifluoroacetic acid, TFA (pH 3.9) and methanol, MeOH solution (20:80, v/v). Afterwards, the separation was achieved at 30ºC with retention times less than 40 min on a Phenomenex Gemini C18 110A column (150x 4.6 mm, 3.0µm particle size) in a single run with a gradient elution of mobile phase consisting of 0.01% TFA pH 4.0 (solvent A) and 100% MEOH (solvent B) at a flow rate of 0.7 mL min^-1 and the detection was performed with diode array (DAD) at different the wavelengths which are obtained maximum signal of analyzed vitamins. Identification and quantification of compounds was achieved by comparing their retention times and peak areas with those of the vitamin standards. The lowest limit of detection (LOD) and limit of quantification (LOQ) were determined and the accuracy of the method was tested by measuring average recovery