Method modification for liquid chromatographic determination of thiamine, riboflavin, and pyridoxine in medical foods.

A reversed-phased ion pair liquid chromatographic method developed for the simultaneous determination of thiamine (B1), riboflavin (B2), and pyridoxine (B6) in perchloric acid extracts of infant formulas was modified to include medical foods. UV detection of B1 and B2 was replaced by fluorescence detection, which resulted in improved sensitivity and specificity. B1 was detected by fluorescence after conversion to thiochrome by a postcolumn reaction with sodium hydroxide and potassium ferricyanide. The method uses a mobile phase of water, acetonitrile, hexanesulfonic acid sodium salt, ammonium hydroxide, and phosphoric acid adjusted to pH 3.6. The column is a 300 x 3.9 mm Nova Pak C18. Limits of detection were 0.05 microgram/mL for B1 and B2 and 0.01 microgram/mL for B6 by fluorescence detection. The system reproducibility was evaluated by completing 10 repetitive determinations on a medical food that gave a coefficient of variation of 5.9, 6.0, and 10.7% for B1, B2, and B6, respectively. Mean recoveries (n = 10) were 111, 96.3, and 113% for B1, B2, and B6, respectively. The results compared favorably with those by AOAC Official Methods 942.23, 940.33, and 961.15 for B1, B2, and B6, respectively.