Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry.
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 x 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.
Pawlosky
R J
RJ
Food Composition Laboratory Beltsville Human Nutrition Research Center, USDA, Beltsville, Maryland 20705, USA. pawlosky@bhnrc.usda.gov
Flanagan
V P
VP
Pfeiffer
C M
CM
D002138:Calibration; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013763:Tetrahydrofolates / Q000097:blood*
D002247:Carbon Isotopes; D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate