Characterization and quantitative estimation of activated cyclophosphamide in blood and urine

An assay for the characterization and quantitative determination of activated cyclophosphamide has been described. The method is based on high reactivity of the kryptaldehyde group in 4-hydroxycyclophosphamide with mercapto compounds to yield 4-(S-R)mercaptocyclophosphamide derivatives. The activated cyclophosphamide can be converted quantitatively to 4-(S-benzyl)mercaptocyclophosphamide with benzyl mercaptan, and the mercapto derivative can be separated by thin-layer chromatography on silica gel with ethyl acetate:methyl ethyl ketone as solvent. Using this assay, significant levels of activated cyclophosphamide in the blood of mice and rats, as well as of humans, were found to be present after cyclophosphamide application. The characterization of 4-(S-benzyl)mercaptocyclophosphamide formed was confirmed by comparing the thin-layer chromatographic and mass spectrometric data on this derivative obtained from an authentic sample of 4-hydroxycyclophosphamide.