Accounting for an Isobaric Interference Allows Correct Determination of Folate Vitamers in Serum by Isotope Dilution-Liquid Chromatography-Tandem MS

Mild and prolonged oxidative degradation of 5-methyltetrahydrofolate (5-methylTHF) leads to the biologically inactive pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF (MeFox). MeFox and the biologically active 5-formyltetrahydrofolate (5-formylTHF) are isobaric compounds that behave similarly during chromatographic and mass separation, making coelution and misidentification likely. Our published routine liquid chromatography-tandem MS (LC-MS/MS) method did not discern between 5-formylTHF and MeFox, measuring the sum of these compounds at a mass to charge ratio (m/z) of 474→327 as 5-formylTHF. We modified this method to separate MeFox and 5-formylTHF by either chromatography or unique mass transitions and then applied the 2 methods to serum specimens to determine typical concentrations of these compounds. The 2 unique transitions (m/z: 5-formylTHF, 474→299; MeFox, 474→284) showed good sensitivity [limit of detection (nmol/L): 5-formylTHF, 0.21; MeFox, 0.34], selectivity (no interfering peaks), spiking recovery (mean ± SD: 5-formylTHF, 103 ± 3.4%; MeFox, 94 ± 10%), and low imprecision (CV: 5-formylTHF, 3.9% at 2.4 nmol/L; MeFox, 5.1% at 2.9 nmol/L). The mass separation method detected 5-formylTHF in the same specimens as the chromatographic separation method. Analysis of several thousand serum specimens showed that the majority (∼85%) contained MeFox at <3 nmol/L but no detectable 5-formylTHF concentrations, some (∼14%) contained 5-formylTHF at <0.5 nmol/L, and a few specimens contained 5-formylTHF at >1 nmol/L and MeFox at >10 nmol/L. In summary, serum can contain 5-formylTHF high enough to contribute to total folate and contains MeFox that will bias total folate if not appropriately separated. Including measurements of MeFox and 5-formylTHF along with the other folate vitamers will enhance assessments of the association between biologically active folate and health effects.