Analysis of commercial spectinomycin samples with ion-pairing reversed-phase LC coupled with electrospray ionization tandem MS (LC/ESI-MS/MS) indicates that eight additional compounds are present, including actinamine, (4R)-dihydrospectinomycin, (4S)-dihydrospectinomycin and dihydroxyspectinomycin, as well as four new impurities reported, to our knowledge, for the first time. The structures of these compounds were elucidated by comparing their fragmentation patterns with known structures, and NMR was employed to characterize and distinguish (4R)-dihydrospectinomycin and (4S)-dihydrospectinomycin. Identification of dihydrospectinomycin isomers is necessary because (4R)-dihydrospectinomycin is a minor active pharmaceutical ingredient of spectinomycin, whereas (4S)-dihydrospectinomycin is considered to be an impurity (impurity C) by the European Pharmacopoeia (Ph. Eur.).