Identification of new metabolites of ifosfamide in rat urine using ion cluster technique

Metabolism of the anticancer drug ifosfamide was investigated in Sprague‐Dawley rats. Along with four known metabolites, namely N2‐dechloroethylifosfamide, N3‐dechloroethylifosfamide, alcoifosfamide and isophosphoramide mustard, four new urinary metabolites were identified utilizing combined techniques of chemical modification/derivatization, capillary gas chromatography/chemical ionization mass spectrometry (ammonia), deuterium‐labeling/ion cluster analysis and chemical synthesis. Secondary metabolites of N2‐dechloroethyl and N3‐dechloroethylifosfamide formed by 4‐hydroxylation, i.e. 4‐hydroxy‐N2‐dechloroethylifosfamide and 4‐hydroxy‐N3‐dechloroethylifosfamide, respectively, and their subsequent decomposition product, N‐dechloroethyliso‐phosphoramide mustard, were identified. Secondary dealkylation pathways of N2‐dechloroethylifosfamide and/or N3‐dechloroethylifosfamide were also demonstrated through characterization of N2,3‐didechloroethyl ifosfamide. The key active metabolite of ifosfamide, 4‐hydroxyifosfamide, was characterized as a cyanohydrin adduct for the first time.