Characterization of impurities in sisomicin and netilmicin by liquid chromatography/mass spectrometry

The characterization of unknown impurities present in netilmicin and sisomicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. The volatile ion-pairing agent trifluoroacetic acid (TFA) was used for the retention of the main compounds and their impurities on a reversed-phase (RP) C18 column, because they are highly hydrophilic and basic compounds. The method showed good separation between netilmicin and its four potential related substances prescribed in the European Pharmacopoeia, which were identified by comparison of their retention times with those of the reference substances. Furthermore, in total 16 unknown impurities in a netilmicin sample and six in a sisomicin sample with unknown identity were detected. The structures of the unknown compounds were deduced based on comparison of fragmentation patterns with those of the reference substances investigated in LC/MSn experiments by the use of electrospray ion trap mass spectrometry.