Folate stability and method optimization for folate extraction from seeds of pulse crops using LC-SRM MS

Folates are essential co-factors for one-carbon metabolism and are needed in human diets. Pulses, such as common bean, lentil and chickpea contain significant amounts of folates. Ultra-performance liquid chromatography coupled with selective reaction monitoring mass spectrometry was used to study the stability of twelve folate monoglutamates at three pHs (5, 7 and 9) under three temperature treatments (ambient, 37 °C and 100 °C). 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, folic acid, 10-formylfolic acid and 10-methyl folic acid are stable at all conditions studied. Conversely, 5-methyl-5,6-dihydrofolate and 10-formyltetrahydrofolate are very labile, with the former rapidly converting to 5-methyltetrahydrofolate, and the latter mainly converting to 5,10-methenyltetrahydrofolate. Under basic conditions without heating, 5,10-methylenetetrahydrofolate is relatively stable, however, in neutral or acidic conditions it degrades and converts to multiple folate forms, mostly tetrahydrofolate. Eight folates were chosen for optimization of extraction from pulse crop seeds. The selection of enzymes, antioxidants and extraction temperature were systematically investigated using CDC Jade (chickpea) as a standard. The optimized one-step extraction includes matrix effect considerations and provides a more precise and accurate method compared with our previous work, while simplifying extraction procedures. MeFox (pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate), an oxidation product of 5-methyltetrahydrofolate, is detected in significant amounts in all four pulse crops.