title: Measurement of folate in fresh and archival serum samples as p-aminobenzoylglutamate equivalents.
abstract: BACKGROUND
  The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis.
  
  
  METHODS
  Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  
  
  RESULTS
  The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25-96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was < or = 5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay.
  
  
  CONCLUSIONS
  The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.
authors.affiliation: Section for Pharmacology, Institute of Medicine, University of Bergen, Bergen, Norway. rita.hannisdal@farm.uib.no
authors.forename: Rita
authors.initials: R
authors.lastname: Hannisdal
authors.forename: Asbjørn
authors.initials: A
authors.lastname: Svardal
authors.forename: Per Magne
authors.initials: PM
authors.lastname: Ueland
mesh_terms: D000328:Adult; D000368:Aged; D002138:Calibration; D002853:Chromatography, Liquid; D005260:Female; D005492:Folic Acid / Q000097:blood*; D005971:Glutamates / Q000097:blood*; D006801:Humans; D006868:Hydrolysis; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D044967:Serum; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D005971:Glutamates; D005492:Folic Acid; C011398:4-aminobenzoylglutamic acid