SIMULTANEOUS DETERMINATION OF PYRIDOXINE, RIBOFLAVIN, AND THIAMINE IN FORTIFIED CEREAL PRODUCTS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High performance liquid chromatography is used to simultaneously determine pyridoxine, riboflavin, and thiamine in fortified cereal products. The vitamins are extracted into dilute acid, followed by enzymatic treatment of the extract with an amylase preparation. No clean-up or pre-concentration of sample extracts is required. The vitamins are separated from other sample components on a reverse phase (ODS) column, using an ion-pairing mechanism. An acidified methanol/water mobile phase containing hexanesulfonate ion as the ion-pairing agent is used to achieve the separation. Pyridoxine and riboflavin are quantitated by fluorescence detection using a 288 nm excitation wavelength. The fluorescent thiochrome derivative of thiamine is formed by addition of an alkaline ferricyanide solution in a post-column reaction system. Thiamine is quantitated as thiochrome by use of a second fluorescence detector. A column switching technique, utilizing a short 50 mm column in conjunction with a six-port high pressure switching valve and the analytical column, is used to minimize the required chromatographic time. Use of the column switching technique allows highly retained components to be eluted from the short column directly to waste, thereby limiting the required chromatographic time to less than 25 min.;The HPLC technique provides better selectivity for riboflavin than do the wet chemical techniques, as the wet chemical procedures rely on chemical reactions to eliminate interferences, and these reactions may not be totally specific for riboflavin. HPLC determination of thiamine provides separation of thiamine from other reducing substances in an extract, such as reducing sugars produced during extraction of sucrose sweetened products, which would otherwise interfere with thiochrome formation. The improved specificity of the HPLC technique provides greater reliability than can be obtained with the wet chemical procedures. Significant time savings are also obtainable over individual, manual, wet chemical assays.;Simultaneous HPLC determination of pyridoxine, riboflavin, and thiamine provides a practical technique for routine, day-to-day usage in quality control applications.;Detection limits for the analytical procedure are 2 (mu)g pyridoxine/g sample, 1 (mu)g riboflavin/g, and 1 (mu)g thiamine/g. Relative standard deviations of the analytical procedure are 1.66%, 1.51%, and 2.06% for pyridoxine, riboflavin, and thiamine, respectively. Comparison of HPLC analytical results with wet chemical values obtained on a set of fortified cereal product samples resulted in correlations of 0.996, 0.998, and 0.996 for pyridoxine, riboflavin, and thiamine, respectively.