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Improved Folate Extraction and Tracing Deconjugation Efficiency by Dual Label Isotope Dilution Assays in Foods

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journal contribution
posted on 15.02.2012, 00:00 by Sabine Mönch, Michael Rychlik
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, [13C5]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [2H4]-5-methyltetrahydrofolate, [2H4]-5-formyltetrahydrofolate, [2H4]-tetrahydrofolate, [2H4]-10-formylfolate, and [2H4]-folic acid. The [2H4]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [13C5]-pteroylheptaglutamate to the detection tracer [13C5]-folic acid, which was quantified along with unlabeled folic acid using [2H4]-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 .