Stability of dietary 5-formyl-tetrahydrofolate and its determination by HPLC: a pilot study on impact of pH, temperature and antioxidants on analytical results

5-Formyltetrahydrofolate (5-HCO-H(4)folate) is one of the main naturally occurring forms of folic acid in foods, but its determination is complicated because of its high susceptibility to interconversion and oxidative degradation. The present study investigated the stability of 5-HCO-H(4)folate at different pH and temperatures and with different antioxidants to find optimal conditions for protection of 5-HCO-H(4)folate from interconversion and oxidation during sample pre-treatment and analysis by HPLC or LC-MS. The pH of buffers during sample pre-treatment and purification was found to be crucial, especially in the absence of antioxidants. To reduce losses of 5-HCO-H(4)folate during sample pre-treatment, the use of two antioxidants, ascorbic acid in combination with 2-mercaptoethanol or 2,3-dimercapto-1-propanol, and pH close to neutral were found to be of great importance. During the chromatographic analysis, the use of moderate acidic buffers in the mobile phase with pH above 3 and shorter retention times of folates, preferably below 15 min, might minimise the losses of 5-HCO-H(4)folate. The results of the present study clearly demonstrated that the interconversion of 5-HCO-H(4)folate to 5,10-methenyltetrahydrofolate at acidic pH should be taken into account when developing HPLC methods for folate determination.