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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry

Desirée E.C. Smith1 / Robert M. Kok2 / Tom Teerlink3 / Cornelis Jakobs4 / Yvo M. Smulders5






Corresponding author: Dr. Yvo M. Smulders, Department of Internal Medicine, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Phone: +31-20-4444307, Fax: +31-20-4444313

Citation Information: Clinical Chemical Laboratory Medicine. Volume 44, Issue 4, Pages 450–459, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2006.085, April 2006

Publication History

November 8, 2005
January 16, 2006


Background: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest.

Methods: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6 min.

Results: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97–107%. We measured 8–72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327 nmol/L and 23 to 363 nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0–2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53–90% loss of the non-methylTHF fraction.

Conclusion: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.

Keywords: erythrocyte; folate; folate vitamer; liquid chromatography; mass spectrometry

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