INVESTIGATION OF THE SOLVENT pH EFFECT ON SHORT-TERM STABILITY OF WATER-SOLUBLE VITAMINS IN SOLUTION
The aim of this work was to investigate the effect of pH on the short-term stability of both individual water-soluble vitamins and their mixtures, thus characterizing the robustness of the method in relation to the pH of the solvent as one of the validation method parameters. Determination of vitamin concentration was performed by liquid chromatographic method immediately after their dissolution, as well as after exposure for 1, 2 and 3 hours at a temperature of 20ºC. The analysis was performed on a Waters liquid chromatograph equipped with an Alliance 2690 separation module with a PAD 996 diode array detector. Sample separation conditions: Luna Omega Polar C18 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase: a mixture of acetonitrile with 0.05 M phosphate buffer solution, pH 3.0. For the separation of thiamine hydrochloride, nicotinamide, pyridoxine hydrochloride and ascorbic acid (vitamin group I), the ratio of components was 3:97, for the separation of ascorbic acid, riboflavin and cyanocobalamin (vitamin group II) - 15:85. The injection volume was 0.01 ml, the mobile phase flow rate was 1.0 ml/min, the column temperature was 25ºC, and the separation time was 10 minutes. The detection wavelength is 265 nm.
It was found that the decrease in the concentration of vitamins B1, B2, B6, B12 and nicotinamide in mixtures with ascorbic acid was within 10% at all pH values except 9.0. When buffer with this pH was used, the concentration of vitamin B1 decreased by 38 % in mixtures with vitamins B6, C and nicotinamide. Vitamin C was the most sensitive to pH. Thus, the greatest decrease in the concentration of ascorbic acid was recorded with the use of phosphate buffer with pH 5.0 and 7.0, both in group I vitamins and when vitamin C was present in solution without other vitamins. At pH 9.0, the concentration of ascorbic acid decreased in the range of 50-80 %. Ascorbic acid was stable when solvent with a pH of 3.0 was used. This solvent is proposed to be used for further validation of methods for determining water-soluble vitamins in premixes and feed additives.
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