AN IMPROVED SIMPLE AND PRECISE HPLC METHOD FOR DETERMINATION OF FOLIC ACID IN FORTIFIED WHEAT FLOURS AND BREADS WITH FLUORESCENCE DETECTION

Folic acid is one of the important hematopoietic agents necessary for proper
regeneration of the blood forming elements and their functioning. Since humans cannot synthesize
folates, they must be obtained from dietary sources. The most important dietary sources of folates are
fortified foods and cereal products with folate content of 50-200 μg/100 g. In Iran the majority of
people consume bread as an ideal food because of its low price and availability. The objective of this
study was to develop a simple and precise High Performance Liquid Chromatography (HPLC) method
for determination of folic acid in enriched wheat flours and bread.
Materials & Methods: The method combines several procedures including an extraction technique,
oxidation of folic acid to increase fluorescent properties, and an improved HPLC with fluorescence
detection method. The method was performed on a C18 analytical column. The mobile phase
consisted of a mixture of methanol and pure water (20/80).The elute was continuously monitored
using a fluorescence detector (λex 296 nm and λem 440 nm).
Results: The standard curve for folic acid passed through the origin and was linear over the range 0.1
– 8µg/L. The peak of folic acid appeared as sharp with retention times of 1.12 minutes. The intra-day
coefficient of variation was evaluated in the range of 3.09-7.82 µg/L. The Inter day coefficients of
variation for the method ranged between 2.37 and 5.07 µg/L. The accuracy of the method ranges
between 99.2% and 103.7% for intra-day analysis and 98.8% and 106.9% for inter-days analysis. The
limit of determination for folic acid was 0.03µg/L. The mean concentration of folic acid was 102 µg
and 85 µg per 100 gram of flour and bread respectively. The amount of folic acid was decreased
during preparation and fermentation process (16.67%).
Conclusions: The obtained results indicated that this method is a rapid, sensitive, and precise
technique for measurement of folic acid in enriched wheat flours and breads