title: Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay.
abstract: BACKGROUND
  Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum.
  
  
  METHODS
  We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards.
  
  
  RESULTS
  Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF.
  
  
  CONCLUSIONS
  This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. CPfeiffer@cdc.gov
authors.forename: Christine M
authors.initials: CM
authors.lastname: Pfeiffer
authors.forename: Zia
authors.initials: Z
authors.lastname: Fazili
authors.forename: Les
authors.initials: L
authors.lastname: McCoy
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Elaine W
authors.initials: EW
authors.lastname: Gunter
mesh_terms: D001322:Autoanalysis; D002853:Chromatography, Liquid; D005492:Folic Acid / Q000097:blood*; D006801:Humans; D007201:Indicator Dilution Techniques; D002955:Leucovorin / Q000097:blood; D008828:Microbiological Techniques; D011869:Radioligand Assay; D012680:Sensitivity and Specificity; D044967:Serum; D021241:Spectrometry, Mass, Electrospray Ionization; D013763:Tetrahydrofolates / Q000097:blood
chemical_list: D013763:Tetrahydrofolates; D005492:Folic Acid; D002955:Leucovorin; C005984:5-methyltetrahydrofolate