Determination of 4-hydroxycyclophosphamide in plasma, as 2,4-dinitrophenylhydrazone derivative of aldophosphamide, by liquid chromatography.

A reversed-phase liquid chromatographic method to determine the concentration of 4-hydroxycyclophosphamide, a labile cytotoxic metabolite of cyclophosphamide, in plasma is described. In order to stabilize 4-hydroxycyclophosphamide, as well as to increase the selectivity and the sensitivity, a 2,4-dinitrophenylhydrazone derivative was formed. Plasma proteins were precipitated with acetonitrile prior to the derivatization with 2,4-dinitrophenylhydrazine at pH 2. The derivatization was performed at 50 degrees C for 5 min. The chromatographic system consisted of an octadecyl silica column and a mobile phase containing phosphate buffer and acetonitrile. Quantitation was performed using an UV detector operating at 357 nm. Optimal derivatization was obtained by adding 0.2 ml 2,4-dinitrophenylhydrazine (3.8 mg/ml) to 0.5 ml of the deproteinized plasma supernatant. The relative recovery of 4-hydroxycyclophosphamide from plasma is > or = 97%. Concentration levels down to 22 mg/ml of 4-hydroxycyclophosphamide in plasma could be determined with a R.S.D. of about 13%. No degradation of the derivative was observed after 24 h at room temperature. The t1/2 for 4-hydroxycyclophosphamide in blood is ca. 4 min at 37 degrees C, whereas 4-hydroxycyclophosphamide is stable for at least 1 h at 4 degrees C. Application of the method for the pharmacokinetic monitoring of 4-hydroxycyclophosphamide is described.