Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Sep;39(9):e70171.
doi: 10.1002/bmc.70171.

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

Jan Thomann  1   2 , Selina Kraus  1   2 , Livio Erne  2   3 , Severin B Vogt  2   3 , Matthias E Liechti  1   2   3 , Dino Luethi  1   2
Affiliations

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

Jan Thomann et al. Biomed Chromatogr. 2025 Sep.

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.

Keywords: bioanalysis; clinical study; liquid chromatography; mass spectrometry; pharmacokinetics.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Chromatographic separation of ketamine (100 ng/mL), norketamine (100 ng/mL), dehydronorketamine (DHNK; 10 ng/mL), (2R,6R)‐hydroxynorketamine (HNK; 100 ng/mL), ketamine‐d4 (10 ng/mL), and norketamine‐d4 (20 ng/mL) in pooled human plasma.
FIGURE 2
FIGURE 2
Selectivity of ketamine (a), norketamine (b), dehydronorketamine (DHNK; c), and (2R,6R)‐hydroxynorketamine (HNK; d) in human plasma. The chromatograms are displayed as an overlay of seven blank samples (left side, black lines), seven double blank samples (right side, black lines), and seven lower limit of quantification (LLOQ; colored lines) samples.
FIGURE 3
FIGURE 3
Pharmacokinetic profiles of ketamine (a), norketamine (b), dehydronorketamine (DHNK; c), and hydroxynorketamine (HNK; d) in plasma of clinical study participants (n = 6, mean ± SEM) receiving an intravenous infusion of 1.0 mg/min ketamine for 55 min.
See this image and copyright information in PMC

References

    1. Dinis‐Oliveira, R. J. 2017. “Metabolism and Metabolomics of Ketamine: A Toxicological Approach.” Forensic Sciences Research 2, no. 1: 2–10. 10.1080/20961790.2017.1285219. - DOI - PMC - PubMed
    1. Glue, P. , Loo C., Fam J., Lane H. Y., Young A. H., and Surman P.. 2024. “Extended‐Release Ketamine Tablets for Treatment‐Resistant Depression: A Randomized Placebo‐Controlled Phase 2 Trial.” Nature Medicine 30, no. 7: 2004–2009. 10.1038/s41591-024-03063-x. - DOI - PMC - PubMed
    1. Harun, N. , Anderson R. A., and Cormack P. A.. 2010. “Analysis of Ketamine and Norketamine in Hair Samples Using Molecularly Imprinted Solid‐Phase Extraction (MISPE) and Liquid Chromatography‐Tandem Mass Spectrometry (LC‐MS/MS).” Analytical and Bioanalytical Chemistry 396, no. 7: 2449–2459. 10.1007/s00216-009-3404-6. - DOI - PubMed
    1. Hasan, M. , Hofstetter R., Fassauer G. M., Link A., Siegmund W., and Oswald S.. 2017. “Quantitative Chiral and Achiral Determination of Ketamine and Its Metabolites by LC‐MS/MS in Human Serum, Urine and Fecal Samples.” Journal of Pharmaceutical and Biomedical Analysis 139: 87–97. 10.1016/j.jpba.2017.02.035. - DOI - PubMed
    1. Hijazi, Y. , Bolon M., and Boulieu R.. 2001. “Stability of Ketamine and Its Metabolites Norketamine and Dehydronorketamine in Human Biological Samples.” Clinical Chemistry 47, no. 9: 1713–1715. - PubMed

LinkOut - more resources