5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

Abstract

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([ 13C 6]folic acid in capsules and [ 2H 2]folic acid in a drink). [ 13C 5]5-methylTHF and [ 2H 4]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 × 10 −11 (=limit of detection) to 3.2 × 10 −7 mol/L and for folic acid over the range 5 × 10 −10 (=limit of detection) to 4.5 × 10 −8 mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [ 13C 6] and [ 2H 2] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8 h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.