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Two structural isomers of cefotiam in cefotiam hydrochloride for injection were observed, and the structures of the isomers were determined by mass spectrometry and various 1D and 2D NMR techniques. The thermo-isomerization mechanism of cefotiam was also discussed. Thermo-isomerization occurred not only in cefotiam but also in cephalosporins containing a 1-alkyl-1H-tetrazole-5-thiol side chain at C-3. Furthermore, the toxic effects of the two impurities of cefotiam hydrochloride were predicted and it is thought that they could be more toxic than cefotiam. The results reported in this article may be important for quality control and stability studies of this class of drugs.