Quantification of Isotope-Labeled and Unlabeled Folates and Folate Catabolites in Urine Samples by Stable Isotope Dilution Assay

Abstract

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 [C-13(5)]-labeled folates from a test dose when performing bio-availability 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, [H-2(4)]-p-aminobenzoyl glutamate, [H-2(4)]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate were synthesized. The synthesis of the [H-2(4)] -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 [C-13(5)]-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 [H-2(4)]-p-aminobenzoyl glutamate, [H-2(4)]-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.

Keywords

internal standard; [H-2(4)]-p-aminobenzoyl glutamate; [H-2(4)]-p-acetamidobenzoyl glutamate; LC-MS/MS; stable isotope dilution assay; dual-label design

Published in

International Journal for Vitamin and Nutrition Research
2013, volume: 83, number: 2, pages: 112-121
Publisher: VERLAG HANS HUBER

SLU Authors

• Öhrvik, Veronica

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Witthöft, Cornelia

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

Global goals (SDG)

SDG2 Zero hunger

UKÄ Subject classification

Molecular Biology
Analytical Chemistry
Biochemistry

Publication identifier

• DOI: https://doi.org/10.1024/0300-9831/a000152

Permanent link to this page (URI)

https://res.slu.se/id/publ/55961