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.

Sarcosine and other metabolites along the choline oxidation pathway in relation to prostate cancer—A large nested case–control study within the JANUS cohort in Norway

“…22 pABG and the above mentioned biochemical markers were stable during sample storage, 23,24 except serine concentration which slightly increased over time. 24 Methylene-tetrahydrofolate reductase (MTHFR) 677C!T (rs1801133) genotypes were determined using matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry.…”

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum

“…We compared the total concentration of folate in 168 serum samples measured by this LC-MS/MS method with the concentrations measured by a microbiologic method and a newly developed pABG assay (34 ). These comparisons gave a correlation of r 2 ϭ 0.92 and r 2 ϭ 0.93, respectively (Fig.…”

Assessment and interpretation of micronutrient status during pregnancy

“…However, recent innovations using liquid chromatography-MS-MS have simplified this process greatly, such that high-volume analysis of urinary and serum p-aminobenzoylglutamate and acetamido-benzoylglutamate may be both possible and cost effective in the future (154,155) . Analysis of p-amino-benzoylglutamate may also have potential as a biomarker of folate status in degraded blood samples (155) . Measurement of folate turnover is not a measurement of folate status per se.…”