Development and validation a LC–MS/MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma: Application to a bioequivalence study

•Simultaneous determination of agomelatine and its metabolites in human plasma by LC–MS/MS.•Simple protein precipitation procedure was employed.•Wide linear ranges for the target analytes were obtained.•Successfully applied to a bioequivalence study in healthy Chinese volunteers.

A novel sensitive and selective LC–MS/MS method for the determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma was developed and validated. After simple protein precipitation, the analytes were separated on a Phenomenex ODS3 column (4.6 × 150 mm, 5 μm, Phenomenex, USA) with mobile phase consisted of methanol and 5 mM ammonium formate solution (containing 0.2% formic acid) at a ratio of 70:30 (v/v) with a flow rate of 0.8 mL/min. The MS acquisition was performed in multiple reactions monitoring (MRM) mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.1, m/z 230.1 → 171.1, m/z 260.1 → 201.1 and m/z 180.1 → 110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and internal standard (phenacetin), respectively. The method was validated for specificity, linearity and lower limit of quantification, precision and accuracy, extraction recovery, matrix effect and stability. The calibration curves for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma were linear over concentration ranges of 0.0457–100 ng/mL, 0.1372–300 ng/mL and 0.4572–1000 ng/mL, respectively. Intra- and inter-day precisions and accuracies data met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method has been successfully applied to a bioequivalence study in healthy Chinese volunteers.