Abstract: AimTo study the dissociation pathways of aminoglycoside antibiotics. MethodsIn positive mode, eight aminoglycoside antibiotics were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. ResultsIt was demonstrated that the eight aminoglycoside antibiotics gave abundant product ions at m/z 322 (gentamicin, micronomicin and sisomicin), m/z 350 (etimicin, netilmicin and vetilmicin) and m/z324 (kanamycin and tobramycin) by loss of the C-ring (amino-α-D-glucopyranose) in MS² full scan mode. In MS³ full scan mode, the prominent fragmentation ions at m/z 163 as well as m/z191 were formed from the fragmentation ions atm/z 322, m/z350 and m/z324 by loss of the A-ring (amino-α-D-glucopyranose), separately, while the characteristic fragmentation ions at m/z 160 as well as m/z162 were formed from m/z 322, m/z350 and m/z324 by loss of the B-ring (2-deoxy-D-streptamine), separately. ConclusionThe structural information was obtained via collision-activated dissociation and these characteristics are applicable to the structural elucidation and quantitative analysis of aminoglycoside compounds.