Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry.

This work reports the use of reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry for the simultaneous profiling of folate-based metabolites including natural folates, their polyglutamatyl derivatives and their biosynthetic precursors in plant and animal tissue. A simple sample preparation method, using 0.1% citric acid and ascorbic acid in ice-cold methanol, was used to extract and stabilise the folates, and three internal standards were used. Chromatography was on a C18 column using slow gradient elution with a mobile phase consisting of methanol/water with 5 mM dimethylhexylamine. Mass spectrometric detection was performed by multiple reaction monitoring in seven separate time windows in negative ion mode over the 25 min run time. Full, quantitative analysis was obtained for 16 folates and a 'semi-quantitative' analysis was possible for all other folates with up to eight conjugated glutamate residues by reference to structurally related calibration standards. The precision, accuracy and recovery of the method were generally within the accepted guidelines for a quantitative bioanalytical method and the method was linear over the range 0.2 to 10 ng of individual folate per sample. The method was applied to profile mono- and polyglutamated tetrahydrofolates (including subcellular analysis) in a range of plant species, including Arabidopsis, spinach, Brassica and wheat; the technique was also successfully applied to the profiling of folates in mouse tissue. Copyright (c) 2005 John Wiley & Sons, Ltd.