Folate determination in cereal-based foods by high-performance liquid chromatography

By federal regulation and beginning January 1, 1998, folic acid must be added to bread and cereals because of its multiple health-related benefits. Therefore, there is a pressing need for a reliable method for rapid, routine determination of folates in fortified cereal products in replacement of the lengthy standard microbiological method. A reversed-phase ion-pair HPLC method was developed and evaluated for achieving this purpose. Added folic acid in vitamin fortified cereal-based foods was extracted using an $\alpha$-amylase preparation for digestion of starch in the sample matrix. Added folic acid was determined within 15 min using a $\rm C\sb{18}$ column and a buffered methanol/water mobile phase containing tetrabutylammonium phosphate as an ion-pairing agent. Quantitation was achieved by UV absorption at 280 nm. Determination of endogenous folates was achieved by treating the sample with rat plasma deconjugase that converted polyglutamyl folates to their monoglutamate residues. After sample clean-up by solid phase extraction, the folates were determined using fluorescence detection with an excitation wavelength of 290 nm and emission wavelength of 350 nm or 450 nm depending on the forms analyzed. Four forms of native folates, tetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolate and 5-methyltetrahydrofolate were measured within 31 min. Total endogenous folate levels ranged from 66 to 191 $\rm\mu g/100$ g in fortified breakfast cereals, 35 to 45 $\rm\mu g/100$ g in unfortified breakfast cereals and 55 to 65 $\rm\mu g/100$ g in unfortified wheat flour. A comparison of results between the chromatographic and standard microbiological methods yielded a high correlation (r = 0.993) between the two procedures. The stability and distribution of added and native folates throughout sponge-and-dough bread processing were assessed using the HPLC procedure described above. Data indicated a good stability of added folic acid at the baking stage, where only 20% was lost. Four native folates described above were detected throughout the process. Their levels increased 72% at the sponge stage, probably due to a contribution from the yeast source, then decreased 32% after baking. However, native folates in bread remained higher (15%) than in the flour