Evaluation of impact of different antioxidants on stability of dietary folates during food sample preparation and storage of extracts prior to analysis

A troublesome factor during folate analysis is the instability of folates, especially tetrahydrofolate. This investigation was therefore performed to evaluate the relative effectiveness of four antioxidants (2-mercaptoethanol, dithiothreitol, 2,3-dimercapto-1-propanol, and 2-thiobarbituric acid) as stabilizing agents. The antioxidants were tested in the combination with 2% sodium ascorbate in acetate, phosphate, and HEPES/CHES buffers commonly used to extract folates from food samples. Baker's yeast was chosen as a model food matrix. A validated method based on reversed-phase high performance liquid chromatography with fluorescence and diode array detection was used for folate analysis. We showed that sample handling was of critical importance in folate analysis. Heat treatment, long-term storage, and repeated freeze/thaw cycles could impair the stability of tetrahydrofolate in varying degrees depending on buffers and antioxidants used. 2,3-Dimercapto-1-propanol was better than 2-mercaptoethanol in protecting tetrahydrofolate under heat extraction, long-term storage of food extracts and freezing/thawing. The use of 2,3-dimercapto-1-propanol as stabilizing agent in folate analysis may, therefore, be preferable owing to its lower toxicity and higher protective effectiveness. Preparation of food samples should include as few freeze/thaw steps as possible before analysis to prevent degradation of tetrahydrofolate.