title: Liquid chromatographic assay based on microextraction by packed sorbent for therapeutic drug monitoring of carbamazepine, lamotrigine, oxcarbazepine, phenobarbital, phenytoin and the active metabolites carbamazepine-10,11-epoxide and licarbazepine.
abstract: A new, sensitive and fast high-performance liquid chromatography-diode-array detection assay based on microextraction by packed sorbent (MEPS/HPLC-DAD) is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in human plasma. Chromatographic separation of analytes and ketoprofen, used as internal standard (IS), was achieved in less than 15min on a C18-column, at 35°C, using acetonitrile (6%) and a mixture (94%) of water-methanol-triethylamine (73.2:26.5:0.3, v/v/v; pH 6.5) pumped at 1mL/min. The analytes and IS were detected at 215, 237 or 280nm. The method showed to be selective, accurate [bias ±14.8% (or ±17.8% in the lower limit of quantification)], precise [coefficient variation ≤9.7% (or ≤17.7% in the lower limit of quantification)] and linear (r(2)≥0.9946) over the concentration ranges of 0.1-15μg/mL for CBZ; 0.1-20μg/mL for LTG; 0.1-5μg/mL for OXC and CBZ-E; 0.2-40μg/mL for PB; 0.3-30μg/mL for PHT; and 0.4-40μg/mL for LIC. The absolute extraction recovery of the analytes ranged from 57.8 to 98.1% and their stability was demonstrated in the studied conditions. This MEPS/HPLC-DAD assay was successfully applied to real plasma samples from patients, revealing to be a cost-effective tool for routine therapeutic drug monitoring of CBZ, LTG, OXC, PB and/or PHT.
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Ferreira
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Márcio
authors.initials: M
authors.lastname: Rodrigues
authors.affiliation: CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Oliveira
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Francisco
authors.affiliation: CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Fortuna
authors.affiliation: Neurology Department, Hospital Centre of Cova da Beira, 6200-251 Covilhã, Portugal.
authors.forename: Luísa
authors.initials: L
authors.lastname: Rosado
authors.affiliation: Neurology Department, Hospital Centre of Cova da Beira, 6200-251 Covilhã, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Rosado
authors.affiliation: CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Amílcar
authors.initials: A
authors.lastname: Falcão
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; CNC-Centre for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal. Electronic address: gilberto@fcsaude.ubi.pt.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Alves
mesh_terms: D000927:Anticonvulsants / Q000097:blood*; D002220:Carbamazepine / Q000031:analogs & derivatives / Q000097:blood; D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D003984:Dibenzazepines / Q000097:blood; D016903:Drug Monitoring / Q000379:methods*; D004852:Epoxy Compounds / Q000097:blood; D006801:Humans; D000077213:Lamotrigine; D000078330:Oxcarbazepine; D010634:Phenobarbital / Q000097:blood; D010672:Phenytoin / Q000097:blood; D011786:Quality Control; D015203:Reproducibility of Results; D014227:Triazines / Q000097:blood
chemical_list: D000927:Anticonvulsants; D003984:Dibenzazepines; D004852:Epoxy Compounds; D014227:Triazines; D002220:Carbamazepine; D010672:Phenytoin; D000077213:Lamotrigine; D000078330:Oxcarbazepine; C428744:10,11-dihydro-10-hydroxy-5H-dibenz(b,f)azepine-5-carboxamide; D010634:Phenobarbital
keywords: Antiepileptic drugs; Bioanalytical method validation; High-performance liquid chromatography; Human plasma; Microextraction by packed sorbent; Therapeutic drug monitoring