Analysis of Ketamine and Xylazine in Rat Tissues Using the ACQUITY UPLC with 2D Technology

Importance of the Topic

Ketamine and xylazine are common veterinary anesthetics that have seen rising illicit use as recreational and date-rape drugs. Detecting these compounds at trace levels in complex solid tissues is crucial for forensic and toxicology laboratories. Traditional sample preparation methods are laborious and time-consuming, motivating the development of faster, high-sensitivity workflows.

Objectives and Study Overview

This work presents a rapid microextraction combined with two-dimensional UPLC (2D UPLC) for quantifying ketamine and xylazine in rat tissues (brain, heart, lung, liver, kidney, spleen). Key goals included optimizing extraction conditions, trap and elute chromatography parameters, and demonstrating robust quantification at low parts-per-trillion levels.

Methodology and Instrumentation

Biological tissues (0.2–0.8 g) were disrupted using high-speed ball-bead homogenization in organic solvent, centrifuged, and the supernatant diluted to <5 % organic content. A mixed-mode SPE (Oasis MCX) protocol provided efficient cleanup without evaporation to dryness. Chromatography employed a trap & elute 2D UPLC setup: an Oasis HLB trap (40 mg) at pH 7, eluted with acetonitrile at pH 3 onto a BEH C18 analytical column, using water/0.5 % formic acid and acetonitrile/0.5 % formic acid gradients. MS/MS detection on Xevo TQ-S in ESI+ mode used two optimized MRM transitions per analyte.

Used Instrumentation

• ACQUITY UPLC with 2D Technology (Trap & Elute configuration)
• Xevo TQ-S tandem quadrupole MS
• Oasis HLB and MCX SPE cartridges
• ACQUITY UPLC BEH C18 column
• MassLynx 4.1 software

Main Results and Discussion

The optimized protocol achieved a method limit of detection of 50 ppt and linear calibration from 0.05 to 10 ppb (R² > 0.998). Extraction recoveries in calf liver exceeded 90 % for both ketamine and xylazine using 4 mL acetonitrile. Matrix effects in liver extracts were 10–40 %, compensated by deuterated internal standards. Quantification of nine rat tissue cases demonstrated consistent detection and quantification across all organs, with clean baselines and stable peak shapes over >1000 injections.

Benefits and Practical Applications

• Total sample preparation in <30 min without evaporation steps
• Fast homogenization (90 s) and high-throughput SPE loading
• Ppt-level sensitivity suitable for forensic casework
• Robust method with minimal matrix interferences

Future Trends and Potential Applications

Advances could include expanding the workflow to additional drug classes, integrating automated on-line SPE, coupling with high-resolution mass spectrometry, and miniaturizing extraction protocols for limited tissue samples. Emerging trap chemistries and faster column technologies will further reduce cycle times and enhance sensitivity.

Conclusion

The combined microextraction and 2D UPLC approach offers a rapid, sensitive, and robust solution for trace analysis of ketamine and xylazine in solid tissues. It streamlines sample preparation, achieves low-ppt detection, and provides reliable data for forensic and biomedical applications.

References

1. Mallet CR, Botch-Jones S. Two-dimension chromatography for trace analytes in tissues. J Anal Toxicol. 2016;1–11.
2. Mallet CR. Multi-Dimensional Chromatography Compendium: Trap & Elute vs At-column Dilution. Waters Corp. 2015;720005339EN.
3. Mallet CR. Analysis of Pharmaceuticals and Pesticides in Water Using ACQUITY UPLC with 2D Technology. Waters Corp. 2014;720005339EN.