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Isolation and mass spectral identification of blood metabolities of cyclophosphamide: Evidence for phosphoramide mustard as the biologically active metabolite
Author(s) -
Struck Robert F.,
Kirk Marion C.,
Witt Maxie H.,
Russell W. Laster
Publication year - 1975
Publication title -
biomedical mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0306-042X
DOI - 10.1002/bms.1200020109
Subject(s) - metabolite , chemistry , cyclophosphamide , chloroform , active metabolite , pharmacology , chromatography , nitrogen mustard , in vivo , thin layer chromatography , biochemistry , medicine , biology , chemotherapy , microbiology and biotechnology
Thin‐layer chromatography and mass spectrometry were used to isolate and identify cyclophosphamide metabolites present in blood of mice. Blood was removed 5, 15, 30 and 45 minutes after intraperitoneal administration and extracted with chloroform followed by methanol. Thin‐layer chromatography of the two extracts and the residual solid with or without prior methylation, collection of resulting alkylating components, determination of radioactivity and mass spectral analysis, served to identify cyclophosphamide, 4‐ketocyclophosphamide, alcophosphamide, N ‐dechloroethylcyclophosphamide, carboxyphosphamide, phosphoramide mustard and nor‐HN2. The absence of detectable levels of 4‐hydroxycyclophosphamide or aldophosphamide in the blood of cyclophosphamide‐treated mice suggests that cyclophosphamide is converted rapidly in the liver to carboxyphosphamide, 4‐ketocyclophosphamide, phosphoramide mustard and nor‐HN2. Direct administration of synthetic 4‐hydroxycyclophosphamide to mice and extraction of blood with chloroform demonstrated the recovery of this metabolite in vivo . Analysis of extracts of blood from mice treated with phosphoramide mustard indicated the presence of nor‐HN2, 3‐(2‐chloroethyl)‐1,3‐oxazolidin‐2‐one and unchanged drug. Consideration of blood levels, cytotoxicity and alkylating activity of metabolites identified in, or inferred from, this study, implicates phosphoramide mustard as a leading candidate for the biologically active form of cyclophosphamide.