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

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


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.

Publication types

  • Validation Study

MeSH terms

  • Cell Line
  • Chemistry Techniques, Analytical / methods*
  • Chemistry Techniques, Analytical / standards
  • Chromatography, High Pressure Liquid*
  • Lamotrigine
  • Limit of Detection
  • Phenytoin / analysis*
  • Phenytoin / isolation & purification
  • Phenytoin / metabolism
  • Reproducibility of Results
  • Triazines / analysis*
  • Triazines / isolation & purification


  • Triazines
  • Phenytoin
  • Lamotrigine