Simple and rapid UPLC-MS/MS method for quantification of entecavir in human plasma: Application to a bioequivalence study .

Abstract

A simple and fast ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated to determine entecavir in human plasma with the stable isotopically labeled internal standard entecavir-¹³C₂¹⁵N. Samples (100µL each) were pretreated by protein precipitation with methanol, and then separated on an ACQUITY UPLC BEH C₁₈ analytical column (2.1 × 50mm, 1.7µm) with a simple isocratic elution. The detection was operated by a positive ionization electrospray mass spectrometry in multiple reaction monitoring mode. The method had a short chromatographic run time of 2minutes, and obtained sharp peaks of entecavir and the internal standard. Good linearity was found within 0.1-20ng/mL. The intra- and inter-day precision and accuracy met the acceptance criteria, and no matrix effect was observed. This method was successfully applied in a bioequivalence study of two kinds of entecavir tablets in healthy Chinese volunteers. And the results showed that no significant differences were found between the test and reference preparations in pharmacokinetic parameters (p>0.05) by ANOVA. The 90% confidence intervals for the geometric mean ratios (test/reference) of C_max, AUC_0-tlast, and AUC_0-∞ fell within the bioequivalence acceptance criteria (80-125%). No significant difference was found in t_max between the two preparations. The two one-sided t-tests showed that these two products were bioequivalent. .