Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2001
Publication Date: 8/15/2001
Citation: Pawlosky, R.J., Flanagan, V.P., Pfeiffer, C. 2001. Determination of 5-methylahydrofolic acid in human serum by stable-isotope dilution hight performance liquid chromatography mass spectromety. Analytical Biochemistry. 298:299-305. Interpretive Summary: Folates form a group of water soluble vitamin compounds whose chemistries are based on a 4-[(pteridin-6-ylmethyl) amino] benzoic acid structure. These vitamins are essential in many cellular processes including, the synthesis of DNA and in cell replication. The predominate form of the vitamin circulating in human blood is the mono-glutamyl form of 5- methyltetrahydofolic acid (5-MTHFA) and the serum concentration of 5-MTHFA is used, in part, to determine the folate status of an individual. Currently, there is a need to develop specific and quantitative assays for the determination of 5-MTHFA. We have developed a specific and selective method for the analysis of 5-MTHFA in human serum utilizing high performance liquid chromatography - electrospray ionization mass spectrometry with positive ion detection. It is expected that this new method will have a major impact on folate analyses in that it has the potential of becoming part of a reference method that will be used to standardize other folate assays.
Technical Abstract: A stable-isotope liquid chromatography-mass spectrometry (LC/ESI-MS) assay was developed for the quantitative determination of the mono-glutamyl form of 5- methyltetrahydrofolic acid in human serum. Serum samples were spiked with 12.2 ng of the internal standard, 13C5-5- MTHFA that had been labeled on the glutamic acid portion of the molecule. The analyte was extracted from the serum onto a solid-phase cartridge and then eluted with an organic solvent and 40 micro-liters (equivalent to 8% of the serum) was taken for LC-MS analysis. The mass spectrum produced upon collision induced dissociation of the analyte in serum was used to confirm the identity of the 5-MTHFA. Using the standard method of addition of 5-MTHFA to serum, a linear dilution curve was constructed. The precision of the method was 5.3% (CV) based on the analysis of four sample replicates. The method was applied to the analysis of a suite of serum samples that contained 5-MTHFA that had been analyzed using other folate methods. The determinations of 5-MTHFA in these samples using the LC-MS procedure were found to be in good agreement with the other methods.