Proposition for Mechanism of Separation of Folates by HPLC-DAD: Practical Application in New Zealand Spinach (Tetragonia expansa)

A fast and simple method for determination of six folate forms was developed through a study of standards and tested in New Zealand spinach (Tetragonia expansa) samples. A systematic evaluation of the standard solubilization conditions, pH, and molar concentration of the aqueous solvent of the mobile phase was performed and evaluated by means of chromatographic parameters: retention time (t r), capacity factor (k), alpha (α), and resolution (Rs). The optimal separation was achieved using phosphate buffer pH 2.0, 100 mmol L−1 and methanol in the ratio 85:15 isocratic mode using C18 Spherisorb ODS-2, 2.0 × 100 mm, 5 μm column, and DAD detection 200–400 nm. The following k values were obtained: 0.58, 1.17, 1.06, 1.98, 3.03, and 6.05, respectively, to tetrahydrofolate, 5-methyltetrahydrofolate, 10-formyltetrahydrofolate, 5-formyltetrahydrofolate, folic, and pteroic acid and α values of 1.81, 1.10, 1.69, 1.53, and 1.99. The elution order of the six folate forms is explained using the Lewis theory of acids and bases. This method was applied to samples of New Zealand spinach and monitored through chromatographic and spectral parameters obtained in the region of maximum absorption of λ. Moreover, the similarity and the purity of the peaks in standards and samples were investigated. For all folate forms, α and Rs values were above 1, except tetrahydrofolate. The similarity index was above 75 % and a purity above 90 % in samples, except for 10-formytetrahydrofolate, indicating that the method has good selectivity for complex matrices.