Identifying decomposition products in extracts of cellular metabolites

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Eseherichia coli with different methanolmater mixtures, we observed that >= 50% water gave an increased yield of nucleosides and bases compared with <= 20% water, as determined by liquid chromatography-tandem mass spectrometry analysis of the resulting extracts. Spiking of the extracts with isotopelabeled nucleotides revealed, however, that the high yield of nucleosides and bases occurred due to decomposition of nucleotides in the water-rich condition, not due to good extraction. Spiking combined with isotope labeling provides a general approach to detecting decomposition products in extracts of cellular metabolites. For extraction of E coli with methanol:water, cold temperature and a high methanol fraction minimize artifacts due to metabolite decomposition. (c) 2006 Elsevier Inc. All rights reserved.