Evidence for an aldehyde possessing alkylating activity as the primary metabolite of cyclophosphamide.

Two-dimensional thin-layer chromatography was used to resolve cyclophosphamide metabolites present (a) in blood and urine of cyclophosphamide-treated rats and (b) after incuba tion of cyclophosphamide with hepatic microsomes, a hepatic 9000 X g supernatant fraction, or a model oxygenase system. Incubation of cyclophosphamide with rat hepatic microsomes yielded a single metabolite (aldophosphamide) capable of alkylating 4-(/>-nitrobenzyl)pyridine. This metabolite could be trapped as the semicarbazone by the addition of semicarbazide to the incubation mixture. Aldophosphamide was unstable to incubation at 37°for 44 hr, degrading to