The zwitterionic drug 3-methyl-9-(2-oxa-2λ5-2H-1,3,2-oxazaphosphorine-2-cyclohexyl)-3,6,9-triazaspiro[5,5]undecane chloride (SLXM-2) is a novel synthetic compound which has shown anticancer activity and low toxicity invivo. In this study, the various gas-phase fragmentation routes were analyzed by electrospray ionization mass spectrometry (positive ion mode) in conjunction with tandem mass spectrometry (ESI-MSn) for the first time. In ESI-MS the fragment ion at m/z 289 (base peak) was formed by loss of the chlorine anion from the zwitterionic precursor SLXM-2. The fragment ion at m/z 232 was formed from the ion at m/z 289 by loss of 1-methylaziridine. The detailed gas-phase collision-induced dissociation (CID) fragmentation mechanisms obtained from the various precursor ions extracted from the zwitterionic SLXM-2 drug was obtained by tandem mass spectrometry analyses. Copyright © 2010 John Wiley & Sons, Ltd.