Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods

A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [ ¹³C ₅]- pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [ ²H ₄]-5- methyltetrahydrofolate, [ ²H ₄]-5-formyltetrahydrofolate, [ ²H ₄]-tetrahydrofolate, [ ²H ₄]-10- formylfolate, and [ ²H ₄]-folic acid. The [ ²H ₄]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [ ¹³C ₅]-pteroylheptaglutamate to the detection tracer [ ¹³C ₅]-folic acid, which was quantified along with unlabeled folic acid using [ ²H ₄]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0. The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 μg/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast.