Simultaneous Quantification of Ketamine and Its Metabolites in Rat Plasma Using LC-MS/MS: Method Development, Validation, and Application

Ketamine (KE) is a clinically important anesthetic and toxicologically relevant compound that undergoes sequential metabolism to norketamine (NKE) and hydroxynorketamine (HNKE).Because parent-only measurements are insufficient to characterize KE disposition, the simultaneous quantification of KE and its metabolites is required.This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of KE, NKE, and HNKE in rat plasma.Plasma samples were prepared using a simple protein precipitation procedure, followed by LC-MS/MS analysis in multiple reaction monitoring mode, and the method was successfully applied to rat plasma samples.The validation results demonstrate that the method is selective, sensitive, precise, and accurate, with acceptable linearity within a defined calibration range.The method was successfully applied to rat plasma (n = 5-7) collected 2 h after intraperitoneal KE administration.This method is expected to be useful for the integrated profiling of KE and its metabolites in rat plasma for pharmacokinetic and toxicological studies.