Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography

A trienzyme treatment (employing rat plasma folate conjugase, alpha-amylase, and protease), followed by affinity chromatography and reversed-phase HPLC, was evaluated for quantification of folates in unfortified and fortified cereal-grain products. Total folate concentrations determined by HPLC for unfortified white and wheat bread, rice, and spaghetti (21.3 +/- 0.69, 29.8 +/- 1.94, 10.8 +/- 0.57, and 22.3 +/- 1.77 mg/100 g of product, respectively) were close to those determined by microbiological assay. In all four products, 5-methyltetrahydrofolate, 10-formyldihydrofolate, 10-formylfolate, 5-formyltetrahydrofolate, and folic acid were identified. The HPLC method allowed good separation and quantification of these folates in unfortified and fortified cereal-grain products within 33 min. Compared to folate conjugase treatment only, trienzyme treatment led to variable increases of measured total folates in white bread (4%), spaghetti (34%), and rice (33%). It is concluded that (a) trienzyme extraction is applicable to both HPLC and microbiological assays and (b) traditional extraction and folate conjugase treatments are not appropriate for analysis of cereal-grain products. This study also provides new information regarding the distribution of naturally occurring folates in this class of foods.