Two-dimensional thin-layer chromatography was used to resolve cyclophosphamide metabolites present (a) in blood and urine of cyclophosphamide-treated rats and (b) after incubation of cyclophosphamide with hepatic microsomes, a hepatic 9000 × g supernatant fraction, or a model oxygenase system.

Incubation of cyclophosphamide with rat hepatic microsomes yielded a single metabolite (aldophosphamide) capable of alkylating 4-(p-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 two metabolites capable of alkylating 4-(p-nitrobenzyl)pyridine, whereas the semicarbazone derivative was relatively stable under the same conditions. Enzymatic action in the hepatic cytosol converted aldophosphamide to carboxyphosphamide.

One hr after cyclophosphamide injection, carboxyphosphamide and aldophosphamide were present in approximately equal amounts in blood. Urine collected for 3 hr after cyclophosphamide injection contained large amounts of carboxyphosphamide and small amounts of aldophosphamide.

Aldophosphamide was also produced by mouse microsomes and by a model oxygenase system.

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This research was supported by USPHS Grant GM 15477. This is Paper 4 in the series on “Cyclophosphamide Metabolism.”

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