Determination of pyridine and adenine nucleotide metabolites in Bacillus subtilis cell extract by sweeping borate complexation capillary electrophoresis

Abstract

With a growing interest in new areas of bioanalytical research such as metabolome analysis, the development of sensitive capillary electrophoresis (CE) methods to analyze sub-μ M concentrations of analytes in biological samples is required. In this report, the application of CE with sweeping by borate complexation is used to analyze a group of seven pyridine and adenine nucleotide metabolites derived from bacteria Bacillus subtilis cell extracts. Nanomolar (n M) detectability of analytes by CE with UV photometric detection is achieved through effective focusing of large sample plug (∼10% of capillary length) using sweeping by borate complexation method, reflected by a limit of detections ( S/ N=3) of about 2×10 −8 M. Changes in metabolites concentrations were observed in cell extracts when using either glucose or malate as the carbon source in the culture medium. Concentration of pyridine and adenine nucleotides in cell extracts varied widely from 78.6 (±7.6) μ M for nicotinamide–adenine dinucleotide in malate to 0.66 (±0.12) μ M for nicotinamide–adenine dinucleotide phosphate in glucose culture medium. Concentrations of metabolites in a single cell were also estimated at milimolar (m M) level. The method was validated in terms of linearity, sensitivity and reproducibility. The application of CE by sweeping borate complexation allows for sensitive and reproducible analyses of nucleotide metabolites in complex biological samples such as bacteria cell extracts.