Development of a novel tandem mass spectrometry method for the quantification of eszopiclone without interference from 2-amino-5-chloropyridine and application in a pharmacokinetic study of rat.

•Illuminated the instability and controllability of eszopiclone (E-ZOP) in biological matrices because of the hydrolysis at different pH points.•Simplified the method of extraction of E-ZOP from biological matrices, liquid-liquid or solid phase extraction was replaced by direct protein precipitation with acetonitrile.•Monitored hydrolysis production via 2-amino-5-chloropyridine (ACP) throughout the whole HPLC-MS/MS method validation for quantified E-ZOP in rat plasma.•Developed and validated the simple, precise and sensitive HPLC–MS/MS method and successfully applied the method to the determination of E-ZOP in rat plasma.