Determination of cyclophosphamide in whole blood and plasma by reversed-phase high-performance liquid chromatography

A rapid, simple, and sensitive reversed-phase high-performance liquid chromatographic determination of the cytostatic drug cyclophosphamide in whole blood and plasma has been developed. The pre-chromatography isolation of the drug involves salting-out of acetonitrile with simultaneous extraction of cyclophosphamide from whole blood and plasma. A short column packed with 5-micron reversed-phase octadecylsilane (ODS) spherical particles was used with an isocratic elution of 5 mM potassium phosphate (pH 6.80)-acetonitrile (80:20, v/v). The cyclophosphamide was monitored at 190 nm and 0.40-0.002 a.u.f.s. At a flow-rate of 1.0 ml/min, the retention time of cyclophosphamide was ca. 9 min. The completion time for the assay was less than 20 min and the assay had a detection limit of 0.30 microgram/ml. This method was used to determine the stability of cyclophosphamide in plasma at room temperature and at -10 degrees C.