Quantitation of acotiamide in rat plasma by UHPLC-Q-TOF-MS: method development, validation and application to pharmacokinetics.

A novel, sensitive and selective ultra-high-performance liquid chromatography-electrospray ionization mass spectrometry method was developed and validated for the quantification of acotiamide (ACT), a first-in-class drug used in functional dyspepsia, in rat plasma. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract ACT from rat plasma. ACT and an internal standard (mirabegron, IS) were separated on an Agilent poroshell EC C18 column (50 × 3.0 mm, 2.7 µm) using methanol-10 mM ammonium acetate binary gradient mobile phase at a flow rate of 0.4 mL/min over 4 min run time. Detection was performed using target ions of [M + H](+) at m/z 451.2010 for ACT and m/z 397.1693 for IS in selective ion mode. The method was validated in the calibration range of 1.31-1000 ng/mL. All the validation parameters were well within the limits. The method demonstrated good performances in terms of intra- and inter-day precision (3.27-12.60% CV) and accuracy (87.96-104.94%). Thus the present ultra-high-pressure liquid chromatograhy-high-resolution mass spectrometry method for determination of ACT in rat plasma, is highly sensitive and rapid with a short run-time of 4 min, can be suitable for high sample throughput and for large batches of biological samples in pharmacokinetic studies. This method can be extended to measure plasma concentrations of ACT in humans to understand drug metabolism, drug interaction and adverse effects.