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 N 2 ‐dechloroethylifosfamide, N 3 ‐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 N 2 ‐dechloroethyl and N 3 ‐dechloroethylifosfamide formed by 4‐hydroxylation, i.e. 4‐hydroxy‐ N 2 ‐dechloroethylifosfamide and 4‐hydroxy‐ N 3 ‐dechloroethylifosfamide, respectively, and their subsequent decomposition product, N ‐dechloroethyliso‐phosphoramide mustard, were identified. Secondary dealkylation pathways of N 2 ‐dechloroethylifosfamide and/or N 3 ‐dechloroethylifosfamide were also demonstrated through characterization of N 2,3 ‐didechloroethyl ifosfamide. The key active metabolite of ifosfamide, 4‐hydroxyifosfamide, was characterized as a cyanohydrin adduct for the first time.