Analysis of Synthetic Cannabinoids in Urine, Plasma, and Edibles Using Multidimensional UPLC Technology

Importance of Topic

The emergence of synthetic cannabinoids in forensic, clinical, and food matrices poses significant analytical challenges. These compounds can appear at trace concentrations in urine, plasma, and edible products, requiring sensitive, accurate, and rapid methods. Reliable detection supports death investigations, clinical monitoring, and regulatory compliance, underscoring the need for robust sample preparation and high-resolution chromatographic techniques.

Objectives and Study Overview

This study aimed to develop and validate a fast micro-extraction protocol combined with multidimensional UPLC-MS/MS for seven synthetic cannabinoids and their metabolites in urine, plasma, and edible matrices. Key goals included achieving ppt-level detection, minimizing carryover, reducing sample-preparation time to 30 minutes, and ensuring quantitative performance suitable for forensic toxicology.

Methodology and Instrumentation

Sample Preparation:

• Urine and plasma: 2 mL sample protein crash with 2 mL acetonitrile; centrifugation; dilution in 100 mL water; mixed-mode SPE (Oasis MAX) with sequential wash and dual-fraction elution (MeOH/NH4OH for neutral/basic and MeOH/FA for acidic analytes).
• Edibles: 1 g homogenized by heating at 70 °C in 50/50 water/MeOH or ACN; supernatant diluted and processed as above.

Multidimensional Chromatography:

• System: ACQUITY UPLC with 2D Technology in trap-and-elute mode; binary pump for analytical gradient, quaternary pumps for loading and at-column dilution.
• Trap column screening: Oasis HLB, XBridge C8/H LB at pH 3–10; analytical column: ACQUITY UPLC HSS T3 (2.1×50 mm, 1.7 μm).
• Optimized method: 5-min gradient from 5% to 95% MeOH+0.5% FA; flow rate 0.6 mL/min; injection volume 200 μL; trap loading at pH 10 (2% NH4OH) on XBridge C8, elution with MeOH+0.5% FA.

Mass Spectrometry:

• Xevo TQD triple-quadrupole with ESI+; multiple reaction monitoring with two transitions per analyte for quantification and confirmation; capillary voltage 3.0 kV, source 150 °C, desolvation 550 °C.

Key Results and Discussion

The 6×6 evaluation grid identified optimal trap and elution conditions (Method 25) providing Gaussian peaks and minimal breakthrough. Switching to HSS T3 improved peak shape and signal intensity. Carryover tests at 10 ppb showed 10% residue, which was reduced below 50 ppt by adding a 2-min post-run reconditioning step using a 1:1:1 MeOH/ACN/acetone wash.
Solid-phase extraction optimization revealed that mixed-mode anion exchange (Oasis MAX) achieved >90% recovery for both neutral/basic cannabinoids and acidic metabolites when using a pooled MeOH/NH4OH and MeOH/FA elution strategy. Sequential elution studies mapped retention, allowing a simplified wash at 70% MeOH/H2O followed by 100% MeOH elution to capture all analytes in a single fraction.
Validation showed:

• Linearity: 0.05–5 ng/mL (urine/edible) and 0.05–10 ng/mL (plasma) with R2 ≥ 0.993.
• Limits of quantification: 0.005–0.05 ng/mL depending on analyte.
• Recoveries: 75–112% across matrices with matrix effects below 10% (urine, edibles) and 19% suppression in plasma.
• Analysis throughput: >1,000 injections with stable performance.

Forensic case samples (urine) demonstrated detection from 0.01 ng/mL to >190 ng/mL, confirming method applicability.

Benefits and Practical Applications

• Rapid sample preparation: 30-min micro-extraction without evaporation steps.
• High sensitivity: ppt-level quantification suitable for postmortem and clinical investigations.
• Robustness: low carryover, stable multi-injection performance, and streamlined SPE workflow.
• Versatility: compatible with biological fluids and solid edibles through simple homogenization.

Future Trends and Opportunities

The integration of at-column dilution and programmable trap columns paves the way for automated multi-analyte panels in forensic laboratories. Further advancements may include on-line sample cleanup, expanded screening libraries for novel synthetic cannabinoids, and incorporation of high-resolution MS for untargeted metabolite profiling. Miniaturized extraction formats and green solvents could enhance throughput and sustainability.

Conclusion

This application note demonstrates a comprehensive workflow combining micro-SPE and 2D UPLC-MS/MS for the rapid, sensitive, and reliable analysis of synthetic cannabinoids in diverse matrices. The optimized protocol delivers robust performance for forensic toxicology, reducing preparation time while maintaining high accuracy and precision at trace levels.

References

1. The Forensic Toxicology Council. What is Forensic Toxicology? SWGTOX, 2010.
2. Mallet, C.R.; Botch-Jones, S. J. Anal. Toxicol. 2016, 1–11.
3. Mallet, C.R. Multi-Dimensional Chromatography Compendium: Trap & Elute vs AT-column dilution; Waters, 2015.
4. Mallet, C.R. Analysis of pharmaceuticals and pesticides in water using ACQUITY UPLC with 2D Technology; Waters, 2014.