Quantitative analysis of anionic metabolites for Catharanthus roseus by capillary electrophoresis using sulfonated capillary coupled with electrospray ionization-tandem mass spectrometry.

This paper describes a practical method to quantify anionic metabolites using contemporary capillary electrophoresis-electrospray ionization-tandem mass spectrometry (CE-ESI-MS/MS). The use of sulfonated capillary permitted a simultaneous analysis of sugar phosphates, organic acids, nucleotides and coenzyme A compounds with only one CE condition. This capillary also improved the repeatability and sensitivity. MS/MS with multiple reaction monitoring (MRM) detection was utilized to achieve significant selectivity and sensitivity. Under optimized CE-ESI-MS/MS system, the detection limits of 53 metabolites at signal-to-noise ratio of 3 were between 0.040 and 8.8 mumol/l. The relative standard deviations (RSDs) for the majority anionic metabolites were better than 0.5% for migration times, and better than 10.0% for peak areas (n=6). Matrix effects by contaminants in sample solution in CE-ESI-MS/MS analysis were removed dramatically by the sample preparation method with liquid-liquid fractionation and ultrafiltration procedure. Furthermore, the developed method was successfully applied to determine anionic metabolites in a cultured cell of Catharanthus roseus. Accumulation of some metabolites including shikimate, malate, and sedoheptulose 7-phosphate by elicitation of methyl jasmonate was observed. The result would show shikimate, tricarboxylic acid and pentose phosphate pathways were activated. Our method will be useful for detailed analysis of primary metabolism dynamics.