title: Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry.
abstract: A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan. soga@sfc.keio.ac.jp
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Soga
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ueno
authors.forename: Hisako
authors.initials: H
authors.lastname: Naraoka
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ohashi
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.forename: Takaaki
authors.initials: T
authors.lastname: Nishioka
mesh_terms: D000838:Anions / Q000032:analysis; D001412:Bacillus subtilis / Q000737:chemistry / Q000378:metabolism*; D002264:Carboxylic Acids / Q000032:analysis; D002952:Citric Acid Cycle; D019075:Electrophoresis, Capillary; D006019:Glycolysis; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards
chemical_list: D000838:Anions; D002264:Carboxylic Acids