AACC collaborative study of a method for determining total folate in cereal products - Microbiological assay using trienzyme extraction (AACC method 86-47)

Regulations published in 1996 by the U.S. Food and Drug Administration requ ire that folic acid be added to a wide range of enriched cereal-grain produ cts, including breads, rolls and buns, wheat flours, corn meals, rice, nood les, and macaroni. Mandatory folic acid fortification of cereal-grain produ cts has emphasized the need for validated methods for determining total fol ate content in foods, particularly cereal products. When the fortification regulations were finalized, no official methods for folate analysis in cere al-grain products were in place. A microbiological assay protocol utilizing a trienzyme extraction procedure was revised to have the conjugase (gamma- glutamyl-carboxy-peptidase) treatment follow a protease treatment, to inclu de the use of cryoprotected inoculum, and to include the spectrophotometric standardization of the standard and optional use of microtiter plates. The draft protocol was prepared in the AOAC format. Thirteen laboratories part icipated in a collaborative study of 10 required and 10 optional samples of cereal-grain products, including flour, bread, cookies, baking mixes, read y-to-eat breakfast cereals, and snack foods. While the majority of the part icipating laboratories performed the assay using the standard test tube met hod, some used the microtiter plate option. The relative standard deviation between laboratories (RSDR) ranged from 2 to 22% for 16 fortified samples compared with expected values of 11 to 20%. RSDR values were higher (i.e., 28-53%) for four unfortified cereal-grain samples. The Youden pair techniqu e was used to estimate within-laboratory relative standard deviations (RSDr ), which ranged from 2 to 13% for fortified samples and from 14 to 21% for pairs that included at least one unfortified sample. Based on the results o f this collaborative study, the microbiological assay with trienzyme extrac tion is recommended for Official First Action status with AOAC and First Ap proval status with AACC.