Pre-Electrospray Ionisation Manifold Methylation and Post-Electrospray Ionisation Manifold Cleavage/Ion Cluster Formation Observed during Electrospray Ionisation of Chloramphenicol in Solutions of Methanol and Acetonitrile for Liquid Chromatography-Mass Spectrometry Employing a Commercial Quadrupole Ion Trap Mass Analyser

We have observed unusual mass spectra of chloramphenicol (CAP) in solutions of methanol or acetonitrile showing intense ions at m/z 297, m/z 311, m/z 325 and m/z 339. The ions observed were different from those which are traditionally observed in the full scan electrospray ionization (ESI) mass spectra of CAP with ions of m/z 321, m/z 323 and m/z 325. We have evidence to show that this process starts with off-line methylation of CAP in solutions of methanol or acetonitrile to give m/z 339. Investigations using nuclear magnetic resonance spectroscopy showed that there is a methylene group somewhere within the CAP molecule but not attached to any of the carbon atoms when the CAP is dissolved in methanol or acetonitrile before infusion into the mass spectrometer. The possible locations of attachment were speculated to be the electronegative atoms apart from the chlorine atoms due to valence considerations. The methylene group is attached to the nitrogen atom and forms a bond as observed in the MS/MS spectra of m/z 297, m/z 311, m/z 325 and m/z 339 which give m/z 183 as the base peak in all cases. Further experiments showed that there is cleavage of the methylated CAP molecule followed by cluster ion formation involving addition of methylene groups to the CAP fragment with m/z 183 to produce ions of m/z including 297, 311, 325 and 339. This process occurs in the mass spectrometer in the region housing the tube lens and is triggered when the ions are accelerated through this region by application of a negative tube lens offset voltage. This region affords collision of the charged droplets with a collision gas in this case nitrogen to strip the droplets of their solvent molecules. Experiments to follow the intensities of m/z 183, m/z 311, m/z 321, m/z 323, m/z 325 and m/z 339 as the tube lens offset voltage was varied were done in which the intensities of m/z 311, m/z 325 and m/z 339 were observed to be at their peak when the tube lens offset voltage was set at −40 V. When the tube lens offset voltage is swung to +40V, thus decelerating the ions through the capillary skimmer region via the tube lens, the traditionally observed spectra with m/z 321, m/z 323 and m/z 325 were observed.