Article ID Journal Published Year Pages File Type
1212773 Journal of Chromatography B 2016 9 Pages PDF
Abstract

•Chiracel OD-R column resolved derivatized 4-hydroxycyclophosphamide enantiomers.•Linearities were 5–5000 ng of each 4-hydroxycyclophosphamide enantiomer/mL plasma.•Pharmacokinetics of 4-hydroxycyclophosphamide was enantioselective in human plasma.

Cyclophosphamide (CY) is one of the most common immunosuppressive agents used in autologous hematopoietic stem cell transplantation. CY is a prodrug and is metabolized to active 4-hydroxycyclophosphamide (HCY). Many authors have suggested an association between enantioselectivity in CY metabolism and treatment efficacy and/or complications. This study describes the development and validation of an analytical method of HCY enantiomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) that can be applied to pharmacokinetic studies, filling this gap in the literature. HCY enantiomers previously derivatized with phenylhydrazine were extracted from 200-μL plasma aliquots spiked with antipyrine as internal standard and a mixture of hexane and dichloromethane (80:20, v/v) was used as the extraction solvent. The derivatized HCY enantiomers were resolved on a Chiracel® OD-R column using water:acetonitrile:formic acid (55:45:0.2, v/v) as the mobile phase. No matrix effect was observed and the analysis of HCY enantiomers was linear for plasma concentrations of 5–5000 ng of each enantiomer/mL plasma. The coefficients of variation and inaccuracy calculated in precision and accuracy assessments were less than 15%. HCY was stable in human plasma after three successive freeze/thaw cycles, during 3 h at room temperature, and in the autosampler at 4 °C for 24 h after processing, with deviation values less than 15%. The method was applied to evaluate the kinetic disposition of HCY in a patient with multiple sclerosis who was pretreated with intravenous racemic CY for stem cell transplantation. The clinical study showed enantioselectivity in the pharmacokinetics of HCY.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
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