2-μm fused-core column ultra-high-performance liquid chromatography/tandem mass spectrometric determination of donepezil in human plasma: Application to a bioequivalence study

Abstract

Ultra-high-performance liquid chromatography (UHPLC) hyphenated to tandem mass spectrometric methods using the dimethylpentafluorophenylpropyl stationary phase with polar and nonpolar characteristics of a 2-μm fused-core silica particle packing were evaluated to perform efficient separation for the analysis of donepezil in human plasma. The fused-core silica particle design providing the shorter diffusional mass transfer path for the analytes is less affected in resolving power by increasing mobile-phase velocity for faster chromatographic resolution between the administered compound and multiple interfering peaks from the extracted quality control, calibration standard and study samples following simple protein precipitation extraction procedures. In this work, two UHPLC-MS/MS approaches requiring 1.2 min per sample run time and neither expensive ultra-high-pressure instrumentation nor new laboratory protocols were applied and compared for determination of donepezil in human plasma at sub-nanograms per milliliter region to support a randomized, crossover bioequivalence (BE) study in which healthy volunteers each received a single oral dose of the test and reference formulations of 10 mg donepezil hydrochloride. The column performance in terms of chromatographic separation efficiency, peak asymmetry and resolution and retention time reproducibility was found to be sustainable over a thousand extracted plasma injections. The linear dynamic range was detected over a concentration range of 0.2–50 ng/mL. The intra- and inter-day assay accuracy and precision were within 15% for the analyte in individual biological fluids. Positive correlation coefficients (r) greater than 0.98 and 0.99 for donepezil concentrations in study plasma samplers measured by the proposed UHPLC-MS/MS approaches and another validated HPLC-MS/MS method were observed.