Simultaneous determination of entecavir and lamivudine in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study
The study's aim is to develop and validate a rapid, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM) mode method for the simultaneous determination of entecavir and lamivudine in rat plasma. Several important conditions were optimized systematically: solid-phase extraction (SPE) cartridges were used to extract the analytes from rat plasma; a hydrophilic interaction chromatography (HILIC) column was employed to separate the target compounds; a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was applied to detect the drug. The method, which required a relatively small volume (50 μL) of plasma sample, was linear over the concentration ranges of 0.5–80 ng mL−1 for entecavir and 50–8000 ng mL−1 for lamivudine. The lower limit of quantitation (LLOQ) of entecavir and lamivudine was 0.5 and 50 ng mL−1, respectively. The method was demonstrated with acceptable accuracy and precision, where the intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were within 15%. Furthermore, the results of both matrix effects and the extraction recoveries of this method were excellent. In conclusion, the developed method was successfully applied to the pharmacokinetic study of entecavir and lamivudine in rats following oral administration of them alone or in combination.