Format

Send to

Choose Destination
J Chromatogr Sci. 2016 Sep;54(8):1352-8. doi: 10.1093/chromsci/bmw088. Epub 2016 May 18.

A Rapid and Sensitive HPLC-DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells.

Author information

1
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.
2
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.
3
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.
4
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.

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.

PMID:
27199444
DOI:
10.1093/chromsci/bmw088
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Silverchair Information Systems
Loading ...
Support Center