AbstractA reversed‐phase ion‐pair high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets. HPLC separation of the vitamins was performed on a Hypersil C18 column and detected by ultraviolet absorbance at 280 nm. The use of methanol‐aqueous 0.5% acetic acid solution (18:82, v/v; containing 2.5 mM sodium hexanesulfonate, pH = 2.8) as the mobile phase at a flow‐rate of 1.2 mL/min enables the baseline separation of the four analytes free from interferences with isocratic elution at 30°C. The analysis time was 17 min per injection. The method was linear in the ranges of 5–90, 2.5–90, 5–95 and 25–450 µg/mL for thiamine mononitrate, riboflavin, pyridoxine hydrochloride and nicotinamide, respectively. The average coefficients of variation of within‐ and between‐day assays were 2.2 and 3.6% for thiamine mononitrate, 1.8 and 2.4% for riboflavin, 1.3 and 1.7% for pyridoxine hydrochloride and 1.0 and 1.5% for nicotinamide, respectively. The average recoveries of thiamine mononitrate, riboflavin, pyridoxine hydrochloride and nicotinamide were 97.0, 97.2, 98.9 and 100.4% for the tablets, respectively. The method has been successfully applied to the simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets. Copyright © 2002 John Wiley & Sons, Ltd.