Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution

Cyclophosphamide (CP) and its metabolite, hydroxycyclophosphamide (OH-CP) have been quantitated in mouse plasma and tissue by derivatization combined with liquid chromatography–tandem mass spectrometry (LC–MS–MS). The derivatization was conducted immediately upon sample collection, to trap the OH-CP metabolite intermediate prior to further conversion to phosphoramide mustard or other reaction products. This simple and straightforward derivatization procedure, combined with sample extraction via protein precipitation, allowed quantitation of CP and the oxime derivative of OH-CP in plasma for concentrations ranging from approximately 12.5–3333 ng/ml, and in spleen tissue for concentrations of 1250–50 000 ng/g. The short cycle time (2.5 min) of the LC–MS–MS method allowed high throughput analysis with minimal matrix interference. Mouse plasma levels were quantitated for doses of 40, 65 and 120 mg/kg; spleen concentrations were determined for mice dosed at 120 mg/kg. The CP and oxime plasma levels correlated well with dose amounts. The CP levels in the spleen and plasma were similar while the oxime levels in the spleen were significantly lower than the plasma.