3 ANALYSIS OF WATER-SOLUBLE VITAMINS IN SUPPLEMENT MIXTURES BY HPLC/UV I. Pencheva, M. Georgieva Medical University – Sofia, Faculty of Pharmacy, Department of Pharmaceutical chemistry, Summary: The present study describes HPLC/UV method with varying conditions (mobile phases content, flow rates, solvents) for quantitation of water-soluble vitamins (WSV) in supplement mixtures. The method was optimized in respects of analytical and chromatographic parameters such as retention time, symmetry factors, column efficiency as number of theoretical plates, capacity factors, specificity, repeatability, LoD, LoQ and linearity. The performance of these method distinguished with excellent application in the assay tests of WSVs in supplement mixtures containing Thiamine, Riboflavine, Pyridoxine, Niacinamide and Folic acid. Key words: WSV (water-soluble vitamins); B vitamins, Nicotinic acid, Folic acid, HPLV/UV. Original Articles Introduction The vitamins are an organic group of compounds with diverse biochemical functions. They are essen- tial dietary components, which are needed in rela- tively small amounts to sustain life and good health. Based on their solubility, vitamins are divided into two main categories: water-soluble vitamins (WSV) and fat soluble vitamins (FSV). WSVs include vi- tamin C (ascorbic acid), B 1 (thiamine), B 2 (ribo- flavine), B 3 (niacin, niacinamide), B 5 (pentothenic acid), B 6 (pyridoxine), B 7 (biotin), B 9 (folic acid), and B 12 (cyanocobalamine). Vitamins are present in almost all types of foods, food sources, vitamin supplements, drugs and etc. in different preparations such as single- or multi- vitamin tablets, injections and vitamin-enriched beverages. Certain foods are commercially forti- fied with vitamins and/or other nutritional essentials such as minerals. The European Union has regulations that define lim- its of vitamin dosages for their safe use as food supple- ments. Most vitamins that are sold as food supplements cannot exceed a maximum daily dosage. Vitamin prod- ucts above these legal limits are not considered food supplements and must be registered as prescription or non-prescription (over-the-counter drugs) due to their potential side effects. All vitamins are pharmacopoeia substances and their quality is defined. In this connection an accurate quantitative measurements for vitamins are required to ensure product quality and regulatory compliance as well as to monitor vitamin intake. The major analytical challenges are that they present [1,2] and there is a need to quantitate them in a wide range of biologi- cal matrices, which include both foods and body fluids (for status indices); [3] the concentrations that are present are usually very low, and the ratio to other components, chemically very similar, is small; [4] they may be present in several or many chemically diverse, but biologically interconvert- ible, forms; [5] some of them are labile to heat, ex- tremes of pH, degradative enzymes and soon and [6] there is no single analytical approach that can quantitate all of them together, within a biological matrix. Established methods for vitamin analysis – from literature and pharmacopoeia include spe- cific chemical titrimetric methods, microbiological methods, which are typically designed for single vitamin analysis and are time consuming [7, 8] and chromatographic methods, including gas chroma- tography [9] capillary electrophoresis [10, 11] and liquid chromatography (LC) with different detec- tion [12–23]. LC methods are the most commonly used methods for simultaneous determination of multiple vitamins and for establishing vitamin pro- files in a variety of matrices with various modes of detection [12–15, 18–20]. The aim of present study is to develop and apply optimized HPLC conditions for simultaneous analy- sis of water-soluble B vitamins, Niacinamide and Fo- lic acid in supplement mixture with high precision, accuracy and specificity.