HPLC-UV Estimation of Folic Acid in Fortified Rice and Wheat Flour using Enzymatic Extraction and Immunoaffinity Chromatography Enrichment: An Interlaboratory Validation Study.

Background: In India, fortification of cereals with folic acid has been voluntary for many years. However, The Food Safety and Standards Authority of India's recent Fortification of Foods Regulations (2018) has prompted us to develop and validate a simple analytical method for estimation of folic acid in fortified cereals.

Objective: The aim was to develop and validate a simple and rugged HPLC-UV method for quantitative analysis of folic acid in fortified rice and wheat flour.

Methods: The enzymatic sample extract was diluted with phosphate buffer, centrifuged, filtered, and then passed slowly through an immunoaffinity cartridge for cleanup. Folic acid in the sample extract was retained by the cartridge and subsequently eluted with 30% acetonitrile [+0.2% trifluoroacetic acid (TFA)]. The elute was collected and analyzed by HPLC-UV at 280 nm. The chromatographic separation of folic acid was achieved on an Agilent Poroshell SB-C18 column (3.0 × 100 mm, 2.7 µm) with 0.1% TFA in methanol as mobile phase.

Results: The linearity range of the vitamin was established in the concentration range of 50-800 µg/L, and the regression coefficient was more than 0.999. The LOQ was 5 µg/L. The average spike recovery values of folic acid in rice and wheat flour samples were 90.9 and 80.5%, respectively. The method was subjected to an interlaboratory validation; eight accredited food testing laboratories across India participated in it and resulted in satisfactory z-scores for the reported results.

Conclusions: The method will be useful in regulatory compliance testing of folic acid in fortified cereals and processed products.

Highlights: A sensitive analysis method is reported for estimation of folic acid in fortified rice and wheat flour. The scope, selectivity, repeatability, and reproducibility of the method establishes it as fit for regulatory compliance check purposes.