High-performance liquid chromatographic determination of stabilized 4-hydroxyifosfamide in human plasma and erythrocytes

Abstract A method using reversed-phase high-performance liquid chromatography (RP-HPLC) is described for the measurement of the stabilized activated metabolite of ifosfamide, 4-hydroxyifosfamide (4-OHIF), in human plasma and erythrocytes. Immediately after sample collection and plasma–erythrocyte separation at 4°C, 4-OHIF was stabilized by derivatization with semicarbazide (SCZ). The sample pretreatment involved liquid–liquid extraction with ethyl acetate. RP-HPLC was executed with a C 8 column and acetonitrile–0.025 M potassium dihydrogenphosphate buffer (pH 7.40)–triethylamine (13.5:86:0.5, v/v) as mobile phase. The analyte was determined with UV detection at 230 nm. Complete validation, optimisation and stability studies were performed and the method proved to be specific, sensitive and with a stable analyte in the range of clinically relevant concentrations (0.1–10 μg/ml) after conventional dosing. The lower limit of quantitation was 100 ng/ml using 1.00 ml of sample. Accuracy was between 94.1 and 107.0%. Within-day and between-day precisions were less than 6.2% and 7.2%, respectively. 4-OHIF-SCZ was found to be stable in the biological matrix at −20°C for at least 1 month. A pharmacokinetic study conducted in a patient receiving 9 g/m 2 over 3 days by means of a continuous infusion, demonstrated the applicability of this method.