Determination of 5-Methyltetrahydrofolic Acid in Human Serum by Stable-Isotope Dilution High-Performance Liquid Chromatography–Mass Spectrometry

• Published in: Analytical biochemistry 2001-11, Vol.298 (2), p.299-305
• Main Authors: Pawlosky, Robert J., Flanagan, Vincent P., Pfeiffer, Christine M.
• Format: Article
• Language: English
• Subjects: 5-methyltetrahydrofolic acid; Calibration; Carbon Isotopes; Chromatography, High Pressure Liquid - methods; electrospray mass spectrometry; folate; human serum; Humans; isotope dilution; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tetrahydrofolates - blood
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2001.5394

The need for specific and sensitive methods for the determination of distinct serum folates is of high priority in clinical research settings. A stable-isotope liquid chromatography–mass spectrometry (LC/ESI-MS) assay was developed for the quantitative determination of the monoglutamyl form of 5-methyltetrahydrofolic acid (5-MTHFA) in human serum. Serum samples (0.5 ml) were amended with the internal standard, [5-13C5]MTHFA that had been labeled on the glutamic acid portion of the molecule and allowed to equilibrate. The analyte was trapped onto a solid-phase cartridge and then eluted with the HPLC mobile phase. Forty microliters was taken for LC/ESI-MS analysis using electrospray ionization operated in the positive ion mode. Using the standard method of addition of 5-MTHFA to serum, a linear dilution curve (y = 12.777x − 1.404; range 0.94–97 ng ml−1) was constructed. The precision of the method was 5.3% (CV) based on the analysis of four sample replicates. The mass spectrum produced upon collision induced dissociation of the analyte in serum was used to confirm the identity of the 5-MTHFA. The method was applied to the analysis of a set of serum samples that contained standardized concentrations of 5-MTHFA. The determinations of 5-MTHFA in these samples using the LC/ESI-MS procedure were found to be in good agreement with other folate methods. A highly accurate and specific method for the analysis of 5-MTHFA in serum has been developed utilizing stable isotope dilution mass spectrometry.