Liquid Chromatography–Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum

Abstract

Folate status is associated with several chronic diseases; thus accurate assessment of folate status has become important in the clinical setting and in epidemiological studies. The diversity of folate forms complicates the task of assaying endogenous folate. We developed and validated an assay that measures various forms of folate in addition to folate catabolites in human serum. We added ascorbic acid to serum samples from 168 healthy blood donors and 39 patients with renal failure, and precipitated the proteins with acetonitrile containing (13)C-labeled folate forms as internal standards. The supernatant was evaporated and the analytes redissolved in water. We then used liquid chromatography-tandem mass spectrometry to quantify 5-methyltetrahydrofolate (5mTHF), 4-alpha-hydroxy-5-methyltetrahydrofolate (hmTHF), folic acid (FA), 5-formyltrahydrofolate (5fTHF), p-aminobenzoylglutamate (pABG), and p-acetamidobenzoylglutamate (apABG). Detection limits were 0.07-0.52 nmol/L, and the assay was linear to 140 nmol/L for all analytes. The mean serum folate concentration from 168 blood donors was 22.7 nmol/L, of which 85.8% was 5mTHF, 12.1% hmTHF, 2.1% FA, and 0.0% 5fTHF. In the same individuals, the mean concentrations of pABG and apABG were 0.07 nmol/L and 0.47 nmol/L, respectively. The concentrations of folate catabolites were 22-30 times higher in 39 patients with renal failure. This folate assay correlated well with the microbiologic assay (r(2) = 0.92) and with measurement of serum folate as pABG equivalents (r(2) = 0.93). This method based on liquid chromatography-tandem mass spectrometry measures the most abundant folate species and 2 folate catabolites in human serum.