Quantification of small molecule organic acids from Mycobacterium tuberculosis culture supernatant using ion exclusion liquid chromatography/mass spectrometry

Quantitative analysis of cellular small molecule organic acids of intermediary metabolism can provide critical insight into bacterial metabolic pathways. The concentration of these metabolites in culture supernatant varies at different growth stages or under particular environmental conditions reflecting both the energy and the biosynthetic needs, yielding metabolic information about the microorganism. The method described here utilizes ion exclusion chromatography with formic acid, coupled with a mass-selective detector using selective ion monitoring with negative mode electrospray ionization (SIM ES-), to detect and quantify several small organic acids in culture supernatants. The microM limits of quantitation (LOQs) were found to be 5.5 +/- 0.9 for pyruvate, 7.0 +/- 0.4 for malate, 2.5 +/- 0.5 for succinate, 12.7 +/- 0.8 for lactate, and 6.6 +/- 0.2 for fumarate. The method was used to detect and quantify these acids in the culture supernatants from Mycobacterium tuberculosis. Supernatant samples were spiked with stable-isotope-labeled internal standards, and the organic acids were quantified by isotope ratiometry.