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Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS.

ABSTRACT: Mild and prolonged oxidative degradation of 5-methyltetrahydrofolate (5-methylTHF) leads to the biologically inactive pyrazino-s-triazine derivative of 4?-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.

SUBMITTER: Fazili Z

PROVIDER: S-EPMC5362735 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS.

Fazili Zia Z Pfeiffer Christine M CM

The Journal of nutrition 20121121 1

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 ...[more]

PMID: 23173171

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Project description:The 15N isotope pool dilution (IPD) technique is the only available method for measuring gross ammonium (NH4 +) production and consumption rates. Rapid consumption of the added 15N-NH4 + tracer is commonly observed, but the processes responsible for this consumption are not well understood. The primary objectives of this study were to determine the relative roles of biotic and abiotic processes in 15N-NH4 + sconsumption and to investigate the validity of one of the main assumptions of IPD experiments, i.e., that no reflux of the consumed 15N tracer occurs during the course of the experiments. We added a 15N-NH4 + tracer to live and sterile (autoclaved) soil using mineral topsoil from a beech forest and a grassland in Austria that differed in NH4 + concentrations and NH4 + consumption kinetics. We quantified both biotic tracer consumption (i.e. changes in the concentrations and 15N enrichments of NH4 +, dissolved organic N (DON), NO3 - and the microbial N pool) and abiotic tracer consumption (i.e., fixation by clay and/or humic substances). We achieved full recovery of the 15N tracer in both soils over the course of the 48 h incubation. For the forest soil, we found no rapid consumption of the 15N tracer, and the majority of tracer (78%) remained unconsumed at the end of the incubation period. In contrast, the grassland soil showed rapid 15N-NH4 + consumption immediately after tracer addition, which was largely due to both abiotic fixation (24%) and biotic processes, largely uptake by soil microbes (10%) and nitrification (13%). We found no evidence for reflux of 15N-NH4 + over the 48 h incubation period in either soil. Our study therefore shows that 15N tracer reflux during IPD experiments is negligible for incubation times of up to 48 h, even when rapid NH4 + consumption occurs. Such experiments are thus robust to the assumption that immobilized labeled N is not re-mobilized during the experimental period and does not impact calculations of gross N mineralization.

Dataset Information

Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS.

Publications

Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS.

Similar Datasets

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