Factors controlling the separation of seven water-soluble vitamins on reversed-phase columns were systematically evaluated. Factors studied include both mobile phase constituents and column parameters. Data showed that a mobile phase containing hexanesulfonate (5 mM), methanol (15%), acetic acid (1%), and triethylamine (0.10-0.13%) yielded excellent separations with several C8 and C18 columns. Lowering the methanol concentration in the mobile phase enhanced the resolution of early eluting peaks, while the triethylamine level controlled the peak shape and retention of thiamine. The analytical precision, robustness, and sensitivity of the developed liquid chromatographic (LC) separation were evaluated. The stability of the LC separation was found to be satisfactory for over a 4-month period.