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).
J Chromatogr B Analyt Technol Biomed Life Sci
; 854(1-2): 345-9, 2007 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-17485255
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 (11, 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 (9010, 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.
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Base de dados:
MEDLINE
Assunto principal:
Cromatografia Líquida de Alta Pressão
/
Ciclofosfamida
/
Espectrometria de Massas por Ionização por Electrospray
Tipo de estudo:
Diagnostic_studies
/
Guideline
Limite:
Humans
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article