ANYL 78 |
| The analysis of intracellular folates is fraught with methodological problems that arise from decomposition and inter-conversion of individual molecular species. This is further complicated by the presence of intracellular polyglutamated forms of folate. To overcome these difficulties a stable isotope dilution liquid chromatography tandem mass spectrometry assay coupled with enzymatic digestion has been developed. Cultured EA.hy 926 endothelial cells were disrupted by sonication and incubated at 37 ºC overnight with gamma-glutamyl hydrolase at pH 4.8 in the presence of appropriate C-13-labled analogs as internal standards. The samples were then stored at pH 2 ready for analysis by LC/MS/MS. The concentrations of folic acid, tetrahydrofolic acid (THF), 5-methyl-THF, 5-formyl-THF, and 5,10-methenyl-THF were then quantified. 10-formyl-THF and some of the 5-formyl-THF were converted to 5,10-methenyl-THF under these conditions. From a three-day validation experiment, precision and accuracy of each analyte ranged from 5.1 to 11.0 %. Supported by NIH grant R01 AR47663. Key Words: folate; quantitation; LC/MS/MS; Eahy926 cell.
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General Papers
7:00 PM-9:00 PM, Sunday, 10 September 2006 Moscone Center -- Hall D, Poster
Division of Analytical Chemistry |