Abstract
Cyclophosphamide (CP) is one of the most frequently used anticancer agents. It is a prodrug requiring activation before exerting cytotoxicity. CP is deactivated to 2-dechloroethylcyclophosphamide (2-DCECP) with formation of an equimolar amount of chloroacetaldehyde. The aim of this study was to develop and validate a sensitive and simple assay for 2-DCECP in plasma of patients treated with CP. Sample pre-treatment consisted of solid-phase extraction of 500 microl of plasma over OASIS HLB (1 ml) cartridges with trofosfamide as internal standard. Separation and detection of underivatized 2-DCECP was performed with capillary gas chromatography with nitrogen/phosphorous selective detection. Extraction recovery of 2-DCECP exceeded 87%. No interference from endogenous compounds, other metabolites of CP and frequently coadministered drugs was detected. The assay was linear in the range of 5-5000 ng/ml in plasma. Accuracy, within-day and between-day precision were less than 11% for the complete concentration range. In plasma, 2-DCECP was stable for at least 1 month when kept at -70 degrees C. Analysis of samples from patients treated with CP demonstrated the applicability of the assay. In conclusion, a sensitive and simple assay for 2-DCECP in plasma, which meets the current requirements for bioanalytical assays, was developed.