A validated method for quantifying entecavir in biological matrices and its application in a pharmacokinetic study in...

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method that uses ganciclovir as an internal standard (IS) has been developed and validated for quantifying entecavir in rat and dog plasma. Following solid-phase extraction (SPE), the analytes were separated on an Inertsil® ODS-3 (5 μm, 150 mm × 2.1 mm i.d.) column and analyzed in selected ion monitoring (SIM) mode with a positive electrospray ionization (ESI) source using the [M+H]+ ions, 278.1 for entecavir and 256.0 for ganciclovir. The method was validated over the concentration range of 0.01-9 μg/mL for entecavir. All precisions (RSD) within and between batches were less than 10%, and accuracies ranged from 98.1 to 102.5%. The lower limit of quantification was 0.01 µg/mL. The extraction recovery averaged 93.9-96.7%. The validated method was used for a pharmacokinetic study of entecavir in rats and dogs. The following pharmacokinetic parameters were obtained for rats and dogs, respectively: the area under the plasma concentration vs. time curves from time 0 to 24 h (AUC0-24) were 15.4 ± 4.5 and 23.4 ± 7.2 μg∙h/mL; the mean maximum plasma concentration (Cmax) were 2.4 ± 0.8 and 5.0 ± 0.9 μg/mL; the mean time to reach the maximum plasma concentrations (Tmax) were 1.7 ± 0.7 and 1.5 ± 0.4 h; and the mean elimination half-life (t1/2) were 5.3 ± 1.4 and 3.8 ± 1.3 h.