An analytical platform comprising three LC-ESI-MS/MS methods is presented for qualitative and quantitative profiling of more than 200 intracellular metabolites. Employing a silica based zwitterionic stationary phase in the HILIC mode, in total 223 hydrophilic metabolites can be determined. In particular, amino acids, organic acids as well as nucleotide sugars were found to be well separable and detectable under acidic mobile phase conditions, while in comparison especially phosphates such as nucleotides, coenzymes or sugar phosphates as well as sugars and sugar acids performed better at higher pH. Additionally, 21 less polar analytes turned out to be amenable for separation and analysis on a pentafluorophenyl modified silica stationary phase in RP mode. Solutes were detected by tandem mass spectrometry on a triple quadrupole instrument in the selected reaction monitoring (SRM) mode and specific SRM transitions for 258 metabolites are provided. All three methods were validated with respect to the limit of quantification, linear dynamic range, precision and accuracy. Applicability of the analytical platform was evaluated by analysis of the targeted metabolites in extracts of beta-lactam antibiotics fermentation broths. Thereby, 87 metabolites were determined qualitatively in penicillin fermentation broths, and 94 compounds were found in cephalosporin extracts. In addition, a number of selected metabolites that can be determined by at least two of the presented LC-MS/MS methods was analyzed quantitatively by both, external calibration using pure standards as well as by matrix-matched calibration performing standard addition. Quantitative results obtained with the different methods agreed well, however, for some analytes external calibration was found to be ill-suited due to matrix effects.
2009 Elsevier B.V. All rights reserved.