title: Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study.
abstract: 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 5-5000ng 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 3h at room temperature, and in the autosampler at 4°C for 24h 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.
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Francine Attié
authors.initials: FA
authors.lastname: de Castro
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
authors.forename: Gabriel dos Santos
authors.initials: Gdos S
authors.lastname: Scatena
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Otávio Pelegrino
authors.initials: OP
authors.lastname: Rocha
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Maria Paula
authors.initials: MP
authors.lastname: Marques
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
authors.forename: Quézia Bezerra
authors.initials: QB
authors.lastname: Cass
authors.affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Belinda Pinto
authors.initials: BP
authors.lastname: Simões
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: lanchote@fcfrp.usp.br.
authors.forename: Vera Lucia
authors.initials: VL
authors.lastname: Lanchote
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C012358:4-hydroxycyclophosphamide; D003520:Cyclophosphamide
keywords: 4-hydroxycyclophosphamide; Enantiomers; LC–MS/MS; Metabolism; Pharmacokinetics