A sensitive method for the determination of entecavir at picogram per milliliter level in human plasma by solid phase extraction and high-pH LC–MS/MS

Entecavir is a guanine nucleoside analogue used in the treatment of hepatitis B virus (HBV) infection. In this paper, we describe an LC–MS/MS method that was developed and validated for the quantitation of entecavir in human EDTA plasma with both high sensitivity (lower limit of quantitation (LLOQ) of 5 pg/mL) and a wide concentration range (5000-fold) intended for low dose ascending clinical studies. High enrichment was achieved by taking advantage of the excellent loading capacity and reproducibility of Oasis HLB 96-well solid phase extraction plate, which allowed 1 mL of plasma samples to be processed in two equal sequential loading steps. Lobucavir, a structural analogue, was used as the internal standard. A filtration step following the reconstitution proved to be vital for the method robustness. The analyte and internal standard were separated on an Xterra MS C18 column with a gradient elution and high-pH mobile phases. Analytes were detected by positive ion electrospray tandem mass spectrometry. The high-pH mobile phase provided both excellent analyte on-column retention and peak shape, leading to the desired sensitivity. Validation results show good intra-assay (12.3%CV) and inter-assay (3.1%CV) precisions, and good assay accuracy (±7.6%Dev). Recovery was high (∼80%), however, the large volume of plasma used did result in a considerable matrix effect (∼0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.