title: Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.
abstract: 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.
  
  
  CONCLUSIONS
  This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.
authors.affiliation: Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Desirée E C
authors.initials: DE
authors.lastname: Smith
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kok
authors.forename: Tom
authors.initials: T
authors.lastname: Teerlink
authors.forename: Cornelis
authors.initials: C
authors.lastname: Jakobs
authors.forename: Yvo M
authors.initials: YM
authors.lastname: Smulders
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000737:chemistry*; D005492:Folic Acid / Q000032:analysis* / Q000378:metabolism; D005838:Genotype; D006801:Humans; D013058:Mass Spectrometry; D008722:Methods; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D015203:Reproducibility of Results; D013763:Tetrahydrofolates / Q000032:analysis
chemical_list: D013763:Tetrahydrofolates; D005492:Folic Acid; D042965:Methylenetetrahydrofolate Reductase (NADPH2); C005984:5-methyltetrahydrofolate