Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry

Abstract

The tissue distribution of folate in its numerous coenzyme forms may influence the development of disease at different sites. For instance, the susceptibility of human colonic mucosa to localized folate deficiency may predispose to the development of colorectal cancer. We report a sensitive and robust ultra high-performance liquid chromatography (UHPLC) tandem mass spectrometry method for quantifying tissue H4folate, 5-CH3-H4folate, 5-CHO-H4folate, folic acid, and 5,10-CH+-H4folate concentration. Human colonic mucosa (20–100mg) was extracted using lipase and conjugase enzyme digestion. Rapid separation of analytes was achieved on a UHPLC 1.9-μm C18 column over 7min. Accurate quantitation was performed using stable isotopically labeled (13C5) internal standards. The instrument response was linear over physiological concentrations of tissue folate (R2>0.99). Limits of detection and quantitation were less than 20 and 30fmol on column, respectively, and within- and between-run imprecision values were 6–16%. In colonic mucosal samples from 73 individuals, the average molar distribution of folate coenzymes was 58% 5-CH3-H4folate, 20% H4folate, 18% formyl-H4folate (sum of 5-CHO-H4folate and 5,10-CH+-H4folate), and 4% folic acid. This assay would be useful in characterizing folate distribution in human and animal tissues as well as the role of deregulated folate homeostasis on disease pathogenesis.