Measurement of 4-hydroxycyclophosphamide in serum by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed for the measurement of 4-hydroxycyclophosphamide (4-OH-CP) in human serum, since no such assay has been described to date. In the present procedure, the serum sample was treated with semicarbazide at pH 7.4 to derivatize the 4-OH-CP to its aldophosphamide semicarbazone form. Derivatization was performed at 60 degrees C for 60 min and the product was extracted with ethyl acetate-chloroform (75:25, v/v). The derivatives formed were chromatographed on a C8 reversed-phase column with a mobile phase of 0.025 M phosphate buffer-acetonitrile (18:82, v/v) and a detection wavelength of 230 nm. The limit of detection of the assay was 0.025 mg/l for 1 ml of serum with a signal-to-noise ratio of 2. The between-assay coefficients of variation at concentrations of 0.2 and 0.4 mg/l were 7.7 and 7.0% respectively. The simplicity and specificity of this method make it directly applicable to clinical studies on 4-OH-CP pharmacokinetics.