High-performance liquid chromatography method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin in pharmaceutical formulations using coulometric electrochemical and ultraviolet detection

The method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin by high-performance liquid chromatography (HPLC) with coulometric electrochemical and UV detections is presented. The retention time of vitamins was repeatedly determined by isocratic elution using 0.05 M phosphate buffer-10% methanol and 0.018 M trimethylamine (1 ml min−1, pH 3.55) as mobile phase with the Supelco LC 18 column 5 μm (25 cm × 4.6 mm). The specificity of the method was demonstrated by the retention characteristics, coulometric electrochemical and UV detection. The limits of detection of thiamine, pyridoxine and cyanocobalamin were: 9.2, 2.7 and 0.08 ng/ml, respectively. The method was characterized also by wide concentration range, high sensitivity and good accuracy (99.6-102.7%). The repeatability of the method was evaluated at different level of concentration of vitamins and the relative standard deviation was below 4.5%. The method was successfully applied for the quantification of Vitamins B1, B6 and B12 in pharmaceutical preparations and dietary supplements.