title: Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods.
abstract: 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, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(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 .
authors.affiliation: Technische Universität München, Division BIOANALYTIK Weihenstephan, Alte Akademie 10, D-85350 Freising, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mönch
authors.forename: Michael
authors.initials: M
authors.lastname: Rychlik
mesh_terms: D002247:Carbon Isotopes; D003903:Deuterium; D005492:Folic Acid / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D005504:Food Analysis / Q000379:methods*; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling; D011624:Pteroylpolyglutamic Acids / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D011624:Pteroylpolyglutamic Acids; D005492:Folic Acid; D003903:Deuterium