Quantification of urinary folate catabolites using liquid chromatography-tandem mass spectrometry

Abstract Folate catabolites p-aminobenzoylglutamate (pABG) and p-acetamidobenzoylglutamate (apABG) in human urine result from break-down of endogenous folate pools and are potential biomarkers of folate status. There is growing interest in analysis of these non-invasive indicators of folate status, since widespread diseases such as cancer, arteriosclerosis and dementia may be linked to disturbed availability of folates. Determination of pABG and apABG in human urine is challenging due to their low urinary concentrations and due to interferences with other urinary compounds. To address these analytical difficulties, we developed an improved LC–MS/MS method with chemical derivatization for fast, selective and sensitive quantification of pABG and apABG in human urine. Forming butyl esters of urinary folate catabolites pABG and apABG improves ionization efficiency as well as enables selective chromatographic separation on standard C18 reversed-phase column material. In contrast to some previously proposed methods for folate catabolites, the new method allows precise differentiation of apABG from pABG. Partial degradation of apABG during derivatization is exactly accounted for using a second differentially labeled stable isotope internal standard. This method is highly sensitive and covers the full range of physiologically occurring concentrations (from 2 to 1000 nmol/L), with volume requirements of only 80 μL urine. Method performance has been validated according to widely accepted standard recommendations. Use of two stable isotope-labeled internal standards and qualifier ion monitoring for both analytes ensure correct identification and unbiased quantification. With run times of less than 2.5 min per sample and cost-efficient sample preparation, this method allows exact quantitation of urinary folate catabolites pABG and apABG for large-scale non-invasive screening of folate status in clinical and epidemiological trials.