The use of cyclic voltammetry to study the oxidation of l-5-methyltetrahydrofolate and its preservation by ascorbic acid

► l-5-MTHF is a natural reduced form of folic acid and could be used as food fortificant. ► Equimolar addition of ascorbic acid preserves l-5-MTHF from oxidation. ► It was demonstrated by cyclic voltammetry that l-5-MTHF has two oxidation sites on its molecule. ► A third oxidation peak appeared after 4h under air at +0.89V. ► Full regeneration of oxidised l-5-MTHF occurred with addition of sodium ascorbate. A cyclic voltammetry study of 1mM l-5-methyltetrahydrofolate (l-5-MTHF) was performed in pH 5.5 Britton–Robinson buffer at room temperature to study the stability of l-5-MTHF alone and in combination with ascorbic acid (AA). The degradation of l-5-MTHF and AA over a period of 12h both followed first order reaction kinetics. Using this technique, oxidation peaks of l-5-MTHF were identified at +0.17 and +1.18V, and another oxidation peak appeared after 4h under air at +0.89V. Cyclic voltammetry and HPLC quantification enable us to confirm that l-5-MTHF can be highly preserved by the addition of an equimolar concentration of AA. This treatment was equivalent to a purge of nitrogen used to remove oxygen and thus minimise oxidation of l-5-MTHF when present in aqueous solutions. HPLC confirmed the fact that a full regeneration of oxidised l-5-MTHF occurred with the addition of sodium ascorbate, thus denoting that the redox character of l-5-MTHF can be controlled by the presence of reducing agents. Cyclic voltammetry proved to be a sensitive and accurate method for characterising l-5-MTHF oxidation and potential preservation with ascorbic acid. To our knowledge, this is the first study that has demonstrated the number of oxidation sites on l-5-MTHF.