Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2001
Publication Date: 10/17/2003
Citation: Thomas, P.M., Pawlosky, R.J., Flanagan, V.P. 2003. Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry. Analytical Biochemistry. 298:299-305. Interpretive Summary: Citrus juices supply an important source of natural folates such as 5 methyltetrahydrofolic acid in the diet. A highly specific and quantitative method was developed to determine the concentrations of 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid in orange and grapefruit juice. The method describes how folates were purified (using a solid phase column cartridge) and analyzed (using high performance liquid chromatography-mass spectrometry). Both 5-MTHFA and folic acid were found in a variety of citrus juices that were surveyed. The national brands of orange juice had much smaller amounts of folic acid compared to store brand juices. Nutrition researchers who are interested in calculating the RDA for folates from foods can use this information for determining the Dietary Folate Equivalent (DFE) from orange and grapefruit juices.
Technical Abstract: This report describes a stable isotope liquid chromatography-mass spectrometry (LC-MS) method that was developed for the quantitative determination of 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid in a variety of citrus juices. Folates were extracted from juices and the polyglutamyl side chain of 5-MTHFA was cleaved to the mono-glutamate form using rat plasma conjugase. The folates were purified on a solid phase column cartridge and analyzed by LC-MS with electrospray ionization. The analytes were quantified using the 13C5 analogues of 5-MTHFA and folic acid as internal standards. The relative standard error of the method was 3.35% based on replicate analysis of four samples. This method was applied to the determination of 5-MTHFA and folic acid in a variety of citrus juices obtained from local supermarkets and the results of the analyses are reported here.