Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclop hosphamide metabolite phosphoramide mustard (PM) in plasma was developed an d validated. PM was determined after derivatisation by high-performance liq uid chromatography (HPLC) with ultraviolet detection at 276 mn. Sample pre- treatment consisted of derivatisation of PM with diethyldithiocarbamate (DD TC) at 70 degrees C for 20 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to th e high salt content of the aqueous phase. The HPLC system consisted of a C- 8 column with acetonitrile-0.025 M potassium phosphate buffer, pH 8.0, (32: 68, v/v) as the mobile phase. The entire sample handling procedure, from co llection at the clinical ward until analysis in the laboratory, was optimis ed and validated. Calibration curves were linear from 50 to 10 000 ng/ml. T he lower limit of quantification and the limit of detection (using a signal -to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 mu l of plasma. Within-day and between-day precisions were below 11% over the enti re concentration range and the accuracies were between 100 and 106%. PM was found to be stable at -30 degrees C for at least 10 weeks both in plasma a nd as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in t wo patients receiving 1000 mg/m(2) CP in a 1-h infusion demonstrated the ap plicability of the assay. (C) 2000 Elsevier Science B.V. All rights reserve d.