title: Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography.
abstract: A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70 degrees C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C8 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 collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10,000 ng/ml. The 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 microl of plasma. Within-day and between-day precisions were below 11% over the entire 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 and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1,000 mg/m2 CP in a 1-h infusion demonstrated the applicability of the assay.
authors.affiliation: Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute, Slotervaart Hospital, Amsterdam. apahu@slz.nl
authors.forename: A D
authors.initials: AD
authors.lastname: Huitema
authors.forename: M M
authors.initials: MM
authors.lastname: Tibben
authors.forename: T
authors.initials: T
authors.lastname: Kerbusch
authors.forename: J J
authors.initials: JJ
authors.lastname: Kettenes-van den Bosch
authors.forename: S
authors.initials: S
authors.lastname: Rodenhuis
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000493:pharmacokinetics*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003520:Cyclophosphamide / Q000493:pharmacokinetics*; D006801:Humans; D010752:Phosphoramide Mustards / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D018906:Antineoplastic Agents, Alkylating; D010752:Phosphoramide Mustards; C030090:phosphoramide mustard; D003520:Cyclophosphamide