Simple method based on fluorescent detection for the determination of 4-hydroxycyclophosphamide in plasma

Cyclophosphamide is a prodrug used both as a single drug and in combination chemotherapy. Cyclophosphamide is converted to its active metabolite (4-hydroxycyclophosphamide) by the cytochrome P450 enzymes. A liquid chromatography method including liquid-liquid extraction and protein precipitation in one step was developed to measure 4-hydroxycyclophosphamide in plasma. The 4-hydroxycyclophosphamide was stabilized and converted to a fluorescent dansylhydrazone derivative, which was chromatographed on a reverse-phase column and detected using a spectrofluorometric detector at excitation of 350 nm and emission of 550 nm. The limit of quantitation was 60 ng/mL and the between-day accuracy and precision were less than 9%. The method was applied to the analysis of plasma from patients who had received an intravenous infusion of 1 g/m(2) cyclophosphamide.