Analysis of intracellular metabolites in bacteria is of utmost importance for systems biology and at the same time analytically challenging due to the large difference in concentrations, multiple negative charges, and high polarity of these compounds. To challenge this, a method based on dispersive solid phase extraction with charcoal and subsequent analysis with ion-pair liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for quantification of intracellular pools of the 28 most important nucleotides. The method can handle extracts where cells leak during the quenching. Using a Phenyl-Hexyl column and tributylamine as volatile ion-pair reagent, sufficient retention and separation was achieved for mono-, di-, and triphosphorylated nucleotides. Stable isotope labeled nucleotides were used as internal standards for some analytes. The method was validated by determination of the recovery, matrix effects, accuracy, linearity, and limit of detection based on spiking of medium blank as well as standard addition to quenched Lactococcus lactis samples. For standard addition experiments, the isotope-labeled standards needed to be added in similar or higher concentrations as the analytes. L. lactis samples had an energy charge of 0.97±0.001 which was consistent with literature, whereas some differences were observed compared with legacy data based on 33P labeling.
- Ion-pair reversed phase liquid chromatography
- Isotope dilution tandem mass spectrometry
- Dispersive solid phase extraction
- Lactococcus lactis