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 ≤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.