title: Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS).
abstract: Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol-acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm x 2.1 mm ID, particle size 5 microm) with 1 mM ammonium hydroxide in water-acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 microL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50-5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide.
authors.affiliation: Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. Corine.Ekhart@slz.nl
authors.forename: Corine
authors.initials: C
authors.lastname: Ekhart
authors.forename: Abadi
authors.initials: A
authors.lastname: Gebretensae
authors.forename: Hilde
authors.initials: H
authors.lastname: Rosing
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Rodenhuis
authors.forename: Jos H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: Alwin D R
authors.initials: AD
authors.lastname: Huitema
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: C012358:4-hydroxycyclophosphamide; D003520:Cyclophosphamide