Fluorometric Determination of 4-Hydroxyifosfamide in Blood and Urine

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A sensitive fluorometric method using 3-aminophenol was developed to measure 4-hydroxyifosfamide, the active metabolite of the antitumor agent ifosfamide, in blood and urine. Serum and urine samples were mixed well with four volumes of citric acid-phosphate buffer (pH 3.5) to stabilize the 4-hydroxyifosfamide. After extraction with dichloromethane, the extract containing 4-hydroxyifosfamide was evaporated to dryness under reduced pressure. The residue was dissolved in distilled water, and the reagent solution of 3-aminophenol in 2N hydrochloric acid was added. After the reaction of 4-hydroxyifosfamide with 3-aminophenol, the fluorescence intensity based on the formation of 7-hydroxyquinoline in the reaction mixture was measured by fluorometry. A linear response was obtained in this assay over the ranges of 1-10 and 5-50 nmol/ml in serum and urine, respectively. Ifosfamide and related compounds such as carboxyifosfamide and ifosfamide mustard did not interfere with the assay. The present method was used to determine the amounts of 4-hydroxyifosfamide in blood plasma and urine samples from rats treated with ifosfamide.

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