OPTIMIZATION OF LIQUID CHROMATOGRAPHIC CONDITIONS TO SEPARATE WATER-SOLUBLE VITAMINS

Abstract

Vitamins are widely used in veterinary medicine, in particular, as components of premixes, feeds and feed additives to increase the productivity of farm animals. Most of these feed additives are a complex amino acid and vitamin mixtures (of ten active substances or more) based on the matrix of plant or animal origin, which can be enriched with protein. When the analyst is faced with the difficult task of analyzing such a mixture, it is usually necessary to use several variants of the HPLC method (high-performance liquid chromatography), which increases the time and cost of analysis of the premix or feed additive. The aim of this work was to optimize the validated method of analysis of riboflavin in premixes and feed additives available in the laboratory for simultaneous (during one chromatographic separation) determination of the content of the maximum amount of water-soluble vitamins. A Waters liquid chromatograph equipped with an Alliance 2690 separation module with a PAD 996 diode array detector was used for separation. Water-soluble vitamins were separated on a Luna Omega Polar C18 250 × 4.6 mm, 5 μm chromatographic column. As solvents were used 0.05 M phosphate buffer pH 3.0 and 0.1 M sodium hydroxide solution (for folic acid). The article describes the stages of liquid chromatographic separation parameters optimization of twelve vitamins and vitamin-like substances (thiamine hydrochloride, riboflavin, flavin mononucleotide phosphate, nicotinic acid, nicotinamide, calcuim pantothenate, phosidinobacino acidorinolobin, hydrocyanide). Chromatograms of standard samples solutions are given, which show the specificity of the technique. Isocratic and gradient elution schemes are investigated. Some parameters of chromatographic peaks under conditions of gradient separation of vitamins are estimated. The parameters of the chromatographic peaks do not exceed the limits recommended in the State Pharmacopoeia of Ukraine (SPU 2.0), that will allow to further carry out a more complete validation of the method for quantitative determination of water-soluble vitamins in various complex matrices: injection and oral solutions, feeds and premixes.