The fortification of flour and bread with folic acid and 5-Methyltetrahydrofolate: the development and validation of the UPLC-MS/MS method for quantitative analysis and bioaccessibility studies using Caco-2 cells

摘要

The body of evidence to date demonstrates an inverse correlation between the folic acid intake and risk of neural tube defects affected pregnancies. This has been the basis for the introduction of the mandatory fortification of bread making flour in Australia. The purpose of the fortification program is to reduce the prevalence of neural tube defects in utero. An accurate quantitative assay is needed to measure the actual folic acid levels in fortified flour and commercially produced bread. An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of folic acid and naturally occurring 5-methyltetrahydrofolate in fortified flour and bread. Optimised sample preparation of bread prior to analysis involved addition of stable isotope labelled internal standards, di-enzyme treatments, solid phase extraction using phenyl-selective cartridges and ultra-filtration. Solid phase extraction was developed to avoid the ion exchange sample preparation techniques, which quickly contaminate the mass spectrometer ion source. A simple and fast cold extraction was optimised for flour analysis. The assay is rugged, fast, accurate and sensitive and is applicable to a variety of food matrices, whilst is capable of the detection and quantification of low folate concentrations, as shown in samples obtained from the transport study. The uptake and transport of folic acid or 5-methyltetrahydrofolate added to bread was determined using Caco-2 cells in vitro absorption model. The findings of this study revealed that the folic acid range in Australian fortified bread varieties was 79-110 µg/100 g and in bread premix and flour was 62-212 µg/100 g. The mean 5-methyltetrahydrofolate concentrations in bread and flour samples ranged between 1.3-3.4 µg/100 g and 2-4 µg/100 g, respectively. The experimental data indicated non-compliance of fortified flour with the mandated levels (200 - 300 µg/100 g flour). The results of the transport study suggested that approximately 14 % of folic acid from fortified bread was transported across Caco-2 cells in one hour, while the transport of added 5-methyltetrahydrofolate was approximately 1.7 %. The fortified bread data presented here provides strong argument in favour of proper monitoring of the levels of added folic acid in Australian bread, otherwise the potential health and social benefits of the mandatory fortification may not be fully realised.