Abstract
A highly selective and sensitive method for identification and
quantification of intracellular metabolites involved in central carbon
metabolism (including glycolysis, pentose phosphate pathway and
tricarboxylic acid cycle) by means of liquid chromatography-tandem
quadrupole mass spectrometry (LC-MS/MS) was developed. The volatile ion
pair modifier tributylammonium acetate (TBAA) was employed in the mobile
phase for simultaneously separation of 29 negatively charged compounds
including sugar phosphates, nucleotides, and carboxylic acids on a
common C 18 reversed-phase column. Method validation results displayed
that limits of detection (LODs) calculated according to DIN (German
Institute for Standardization) 32645 are mostly below 60 nM, only with
the exception of pyruvate and malate. The calibration curves showed
excellent linearity mainly over three orders of magnitude with
correlation coefficients R-2 > 0.9982. This LC-MS/MS method was
successfully applied to determine these metabolites in cell extracts of
Escherichia coli. Most of the intracellular metabolites were found
within the detection range and the relative standard deviations of the
measurements were smaller than 5.65\% (n = 5) for a cell extract sample.
(c) 2007 Elsevier B.V. All rights reserved.
Users
Please
log in to take part in the discussion (add own reviews or comments).
