title: A Rapid and Sensitive HPLC-DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells.
abstract: A sensitive and fast high-performance liquid chromatography-diode-array detection assay was developed and validated for the simultaneous quantification of 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), phenytoin (PHT) and lamotrigine (LTG) in samples of cultured HepaRG cells. Chromatographic separation of analytes and internal standard (IS) was achieved in ∼15 min on a C18-column, at 35°C, using acetonitrile (6%), methanol (25%) and a mixture (69%) of water-triethylamine (99.7:0.3, v/v; pH 6.0), pumped at 1 mL/min. The analytes and IS were detected at 215 or 235 nm. Calibration curves were linear with regression coefficients >0.994 over the concentration ranges of 0.1-15 µg/mL for HPPH; 0.15-30 µg/mL for PHT and 0.2-20 µg/mL for LTG. The method showed to be accurate (bias value of ±10.5 or ±17.6% in the lower limit of quantification, LLOQ) and precise (coefficient variation ≤8.1 or ≤15.4% in the LLOQ), and the absolute recovery of the analytes ranged from 62.5 to 96.9%. HepaRG cells have emerged as a very promising in vitro model to evaluate metabolic, drug interaction and/or pharmacokinetic studies, and this methodology will be suitable to support subsequent studies involving the antiepileptic drugs PHT and LTG.
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, Covilhã 6200-506, Portugal CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, 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, Covilhã 6200-506, Portugal UDI-IPG-Research Unit for Inland Development, Polytechnic Institute of Guarda, Guarda, Portugal.
authors.forename: Márcio
authors.initials: M
authors.lastname: Rodrigues
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, Portugal Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra 3000-548, 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, Covilhã 6200-506, Portugal CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-517, Portugal gilberto@fcsaude.ubi.pt.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Alves
mesh_terms: D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D000077213:Lamotrigine; D057230:Limit of Detection; D010672:Phenytoin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D014227:Triazines / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D014227:Triazines; D010672:Phenytoin; D000077213:Lamotrigine