Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6-O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2-C3 and O-C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6"-O-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6-O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.