Analysis of 4-hydroxycyclophosphamide in human blood.

Cyclophosphamide is a prodrug activated by cytochrome P450 isozymes in the liver. The product of hepatic activation of cyclophosphamide is 4-hydroxycyclophosphamide. Previously reported methods for determining 4-hydroxycyclophosphamide were either impractical or unreliable for monitoring infusion pharmacokinetics in conjunction with clinical trials. One procedure in which a fluorescent hydroxyquinoline derivative was prepared from 4-hydroxycyclophosphamide and analyzed by HPLC appeared to work at first, but gradually lost its selectivity due to degradation of the column by the strongly acidic mobile phase. An alternative procedure was developed using a weakly acidic eluent and postcolumn treatment with trifluoroacetic acid. This provided for protonation of the hydroxyquinoline, required for sensitive fluorescence detection, but spared the column. The resulting assay was sensitive, selective, reproducible, and accurate. The method was used to monitor 4-hydroxycyclophosphamide pharmacokinetics during and after 4 day infusions of 1.5 g/m2-day of cyclophosphamide given to three patients. It was also used to measure the time-dependent disappearance of acrolein and 4-hydroxycyclophosphamide added to human blood from healthy donors and that of metabolically derived 4-hydroxycyclophosphamide in the blood of a patient treated with cyclophosphamide. Slower decomposition was observed in the latter two cases than in the blood spiked with acrolein. Reliable data were obtained from > 1000 determinations using the same column without significant degradation of its stationary phase.