Development and full validation of a UPLC-MS/MS method for the simultaneous quantification of esketamine, noresketamine, and hydroxynoresketamine in beagle dog plasma and its application to a pharmacokinetic study
Year: 2026
Type: article
Abstract: Background Esketamine (ESK), the S-enantiomer of ketamine, has garnered significant clinical interest as a rapid-acting antidepressant. Its pharmacokinetic profile is critically shaped by its major metabolites, noresketamine (NESK) and hydroxynoresketamine (HNESK), which may contribute to its overall pharmacological effects. Reliable bioanalytical methods for simultaneous quantification of ESK and these metabolites in preclinical species are essential for translational research. Methods We developed and comprehensively validated a highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the concurrent determination of ESK, NESK, and HNESK in beagle dog plasma. Analyte separation was achieved on a UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) using gradient elution with a mobile phase composed of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4 mL/min, with a total run time of 4.0 min. Methoxyphenamine served as the internal standard. Detection was performed in positive electrospray ionization mode with multiple reaction monitoring, employing specific ion transition pairs for each target compound to guarantee selectivity and accuracy. Sample preparation involved a simple one-step protein precipitation with acetonitrile using only 100 µL of plasma. Results The method exhibited excellent linearity across the tested concentration ranges, with all calibration curves yielding determination coefficients (R 2 ) exceeding 0.999 7. The lower limits of quantification were 1 ng/mL for both ESK and NESK, and 0.5 ng/mL for HNESK. Intra- and inter-batch precision (RSD, %) ranged from 3.17% to 14.22% for ESK, 5.41%–10.94% for NESK, and 4.70%–12.40% for HNESK, while accuracy (RE, %) was within ±15% for all analytes. Matrix effects were well controlled (ME%: 93.27%–104.59%), and mean extraction recoveries exceeded 80.22%. Stability under relevant storage and handling conditions was also confirmed, along with satisfactory dilution integrity for 2-fold dilution. The validated method was successfully applied to a pharmacokinetic study in beagle dogs following a single intramuscular dose of 1.0 mg/kg ESK, providing reliable concentration-time profiles for all three analytes. Conclusion This is the first reported UPLC-MS/MS method enabling simultaneous quantification of ESK, NESK, and HNESK in beagle dog plasma. The assay features a short run time (4.0 min), simple sample preparation, low plasma volume requirement (100 µL), and excellent sensitivity, making it a robust and practical analytical tool for preclinical pharmacokinetic investigations and future mechanistic studies of ESK and its active metabolites.
Source: Frontiers in Pharmacology
Institution: Henan University of Science and Technology
Language: English
Subfield: Analytical Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W7225890818