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Tri-enzyme extraction and microbiological assay of total folate in cereal products: AACC/AOAC
collaborative study report. P. M. KEAGY (1), J. DeVries (2), J. Rader (3), and C. A. Hudson (1). (1)
ARS, USDA, Albany, CA 94710; (2) Medallion Laboratories, Minneapolis, MN 55427; (3) FDA,
Washington DC 20204.
Mandatory folic acid fortification of enriched cereal-grain products has
emphasized the need for validated methods for total folates in foods, particularly cereal products. At present
there is no AACC method for folic acid and the current AOAC method for infant formula does not
incorporate recent improvements in the extraction procedure. The tri-enzyme extraction and
microbiological assay method as used by FDA laboratories was written in AOAC format and sent for
comment to 40 laboratories known to be experienced in folate analysis. The method was revised and 13
laboratories participated in a collaborative study of 10 required and 10 optional samples including flours,
bread, cookies, macaroni, corn meal, baking mixes, breakfast cereals, and a snack food. Based on mean
sample folate concentrations, relative standard deviations between laboratories, RSD (R), were expected to
fall between 11 and 20%. RSD (R) for 16 fortified samples (56 to 1342 micrograms folic acid/100g) ranged
from 2% to 22%. RSD (R) for four unfortified samples (23 to 63 micrograms folic acid /100g) ranged from
28% to 53%. The Youden Pair technique was used to estimate within laboratory relative standard deviations
which ranged from 2 to 13% for fortified samples and from 14 to 21% for pairs which included at least one
unfortified sample.
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