LC–ESI-MS/MS method for the quantification of entecavir in human plasma and its application to bioequivalence study

Abstract: Liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) was used for a quantitative estimation of entecavir (EV) in human plasma using lamivudine (LM) as internal standard (IS). The method herein described is simple, sensitive, and specific. Chromatographic separation was performed on XBridge-C18, 4.6mm×50mm, 5-μm column with an isocratic mobile phase composed of 10mM ammonium hydrogen carbonate (pH 10.5):methanol (85:15 v/v), pumped at 0.3ml/min. EV and LM were detected using proton adducts at m/z 278.1→152.1 and 230.2→112.0 in multiple reaction monitoring (MRM) positive mode. Solid phase extraction method was employed in the extraction of EV and LM from the biological matrix. This method was validated over a linear concentration range of 50.0–20000.0pg/ml with a correlation coefficient (r) ≥0.9983. Intra and inter-day precision of EV was found within the range of 1.2–4.2 for EV and 4.4–4.5 for LM. EV was stable throughout three freeze/thaw cycles, bench top and postoperative studies. This method was successfully used in the analysis of plasma samples following oral administration of EV (0.5mg) in 26 healthy human volunteers. [Copyright &y& Elsevier]