Development and Validation of a Rapid LC–MS/MS Method for Plasma Analysis of Ketamine, Norketamine, Dehydronorketamine, and Hydroxynorketamine

Abstract

Ketamine, a well-established dissociative anesthetic, has recently gained significant attention for its rapid-acting antidepressant effects, particularly in treatment-resistant depression. In this study, we developed and validated a state-of-the-art liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the bioanalysis of ketamine and its metabolites, norketamine, dehydronorketamine (DHNK), and (2R,6R)-hydroxynorketamine (HNK), in human plasma. The method features a small sample volume, a streamlined protein precipitation protocol, and a rapid sample runtime. The mobile phase gradient is composed of an aqueous ammonium hydrogen carbonate solution and pure acetonitrile. Using positive electrospray ionization, linear quantification ranges of 1–1,000 ng/mL were established for ketamine and norketamine, while ranges of 0.25–100 ng/mL for DHNK and 2.5–1,000 ng/mL for (2R,6R)-HNK were achieved. The method demonstrated high accuracy, precision, selectivity, and sensitivity, along with consistent matrix effects, efficient extraction recovery, and analyte stability. Finally, the method was successfully applied to assess the pharmacokinetics of six clinical trial participants. Overall, this LC–MS/MS method offers a robust and efficient approach for the achiral quantification of ketamine and its metabolites in human plasma. Its minimal sample preparation and reduced analytical runtime make it particularly well-suited for clinical studies, drug monitoring, and forensic investigations.