Quantification of isotope-labeled and unlabeled folates and folate catabolites in urine samples by stable isotope dilution assay.
Dual-label stable isotope dilution assays for the simultaneous quantification of isotopologic folates in clinical samples offer the perspective for differentiating between unlabeled folates from endogenous body pools and administered [13C5]-labeled folates from a test dose when performing bioavailability trials. In contrast to intact folates, this methodology could hitherto not be applied to the quantification of the folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate. In this study, [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate were synthesized. The synthesis of the [2H4]-labeled compounds started at unlabeled p-aminobenzoic acid. For the formation of p-acetamidobenzoyl glutamate, p-aminobenzoyl glutamate was acetylated. The new substances were applied successfully in stable isotope dilution assays for the simultaneous quantification of the [13C5]-labeled and unlabeled folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate, along with the predominant folate vitamers in urine. The assays were based on clean-up by strong anion exchange followed by liquid chromatography-tandem mass spectrometry detection. Assay sensitivity was sufficient to detect the folate catabolites in physiologic concentrations. The limit of detection was below 0.4 and 0.3 nmol/100 g for p-aminobenzoyl glutamate isotopologues and p-acetamidobenzoyl glutamate isotopologues in urine, respectively. The successful synthesis of [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate and the implementation of these substances in stable isotope dilution assays allows dual-label designs that provide a more detailed insight into human folate metabolism.
Division BIOANALYTIK Weihenstephan, Research Center for Nutrition and Food Sciences (Z I E L), Technische Universität München, Freising, Germany.
Barbara E
BE
Büttner
Department of Food Science, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Veronica E
VE
Ohrvik
German Research Center for Food Chemistry, Leibniz Institute Freising, Germany.
Peter
P
Köhler
Department of Food Science, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Cornelia M
CM
Witthöft
Division BIOANALYTIK Weihenstephan, Research Center for Nutrition and Food Sciences (Z I E L), Technische Universität München, Freising, Germany, and Chair of Analytical Food Chemistry, Technische Universität München, Freising, Germany.
Michael
M
Rychlik
D002247:Carbon Isotopes; D003903:Deuterium; D005492:Folic Acid / Q000652:urine*; D005971:Glutamates / Q000652:urine; D006801:Humans; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D062366:para-Aminobenzoates / Q000652:urine
D002247:Carbon Isotopes; D005971:Glutamates; D062366:para-Aminobenzoates; C030903:N-(4-acetamidobenzoyl)-L-glutamic acid; D005492:Folic Acid; C011398:4-aminobenzoylglutamic acid; D003903:Deuterium
LC-MS/MS; [2H4]-p-acetamidobenzoyl glutamate; [2H4]-p-aminobenzoyl glutamate; dual-label design; internal standard; stable isotope dilution assay