A Comprehensive, Fast, and Sensitive Method for the Quantitation of Synthetic Cannabinoids using the Elute UHPLC Coupled to an EVOQ LC-TQ MS

Significance of the Topic

The rapid emergence of numerous synthetic cannabinoids poses a significant analytical challenge for forensic and clinical laboratories. These compounds, often full agonists at CB1 and CB2 receptors, can produce severe physiological and psychological effects and are frequently modified to evade legislation. A sensitive, specific, and flexible method for their quantitation in human serum is essential for drug monitoring, driving‐under‐influence cases, and post-mortem investigations.

Objectives and Study Overview

This work aims to develop and fully validate a high-throughput UHPLC–triple quadrupole MS method capable of simultaneous quantification of 99 synthetic cannabinoids in serum at sub-ng/mL levels. Key goals include achieving low limits of detection and quantitation, maintaining specificity amid complex biological matrices, and ensuring adaptability for novel analytes.

Methodology and Instrumentation

• Sample Preparation
  • Serum aliquot (1 mL) alkalinized with pH 10 buffer (0.5 mL).
  • Liquid–liquid extraction performed with two phases of hexane:ethyl acetate (99:1 and 80:20) including deuterated internal standards.
  • Combined extracts evaporated and reconstituted in 100 µL eluent (50:50 mobile phases A:B).
• Liquid Chromatography
  • System: Bruker Elute UHPLC; column: Kinetex C18 (100 × 2.1 mm, 2.6 µm).
  • Mobile phases: A – 1 % ACN/99 % water with 0.1 % formic acid and 2 mM ammonium formate; B – 99 % ACN with same additives.
  • Gradient: 20 % B to 90 % B over 8 min; total run time 12 min; flow rate 500 µL/min; injection volume 10 µL; column oven 40 °C.
• Mass Spectrometry
  • Instrument: Bruker EVOQ Elite triple quadrupole MS; VIP H-ESI positive mode.
  • Collision gas: argon (1.5 mTorr); probe gas, cone gas, and exhaust optimized for sensitivity.
  • MRM transitions: two per analyte, one per internal standard; MRM builder tool used for transition and collision energy optimization.
• Validation
  • Guidelines: German GTFCh and DIN 32645.
  • Parameters: selectivity, matrix effects, recovery, LOD/LOQ, linearity, precision, accuracy.
  • Calibration: seven levels (50 pg/mL–1.25 ng/mL), weighting 1/x²; QC at 0.05, 0.25, 1 ng/mL.

Main Results and Discussion

• Chromatographic separation of 99 cannabinoids achieved in 8.5 min with a 12 min cycle time.
• Selectivity confirmed: no interferences in blank serum for targeted transitions.
• Matrix effects within ±25 % for 89 analytes; recoveries >50 % for 79 analytes.
• LOD <5–10 pg/mL for 89 compounds (highest 50 pg/mL); LOQ ≤30 pg/mL for 94 compounds, set at 50 pg/mL for all.
• Linearity (r² > 0.99) and accuracy within ±15 % for most analytes; six compounds reported semi-quantitatively due to occasional bias.
• Interday and intraday precision RSD <15 % across QC levels.

Practical Benefits and Applications

• High sensitivity enables detection of recent use in forensic and clinical contexts.
• Rapid UHPLC cycle enhances sample throughput in high-volume laboratories.
• MRM builder simplifies inclusion of emerging cannabinoids into the method.
• Applicable to quality control, toxicological screening, driving-under-influence testing, and post-mortem analysis.

Future Trends and Potential Applications

• Integration of isotopically labeled analogs for improved compensation of matrix effects.
• Expansion to additional biological matrices (urine, oral fluid, tissues).
• Real-time data evaluation with automated MRM libraries and AI-driven peak confirmation.
• Adaptation to high-resolution MS to capture untargeted new psychoactive substances.

Conclusion

The presented UHPLC–triple quadrupole MS assay delivers a comprehensive, rapid, and robust solution for simultaneous quantitation of 99 synthetic cannabinoids in serum at sub-ng/mL levels. Its strong performance in sensitivity, selectivity, precision, and adaptability ensures it meets the evolving needs of forensic, clinical, and research laboratories.

Reference

1. Matuszewski, M.L.; Constanzer, C.M.; Chavez-Eng, C.M. Anal. Chem. 2003, 75(13), 3019–3030.
2. DIN 32 645 Nachweis-, Erfassungs- und Bestimmungsgrenze; Beuth Verlag: Berlin, 1994.