SOFT: CONFIRMATION OF CANNABINOIDS IN FORENSIC TOXICOLOGY CASEWORK BY ISOMER-SELECTIVE UPLC-MS/MS

Significance of the Topic

The accurate confirmation of ∆9-tetrahydrocannabinol (∆9-THC) use in forensic urine testing has been challenged by the emergence of cannabidiol-derived isomers such as ∆8-THC and ∆10-THC. Traditional methods targeting only the major metabolite 11-nor-9-carboxy-∆9-THC risk misidentification and interference. An isomer-selective approach is essential to support legal and investigative outcomes.

Objectives and Study Overview

This study presents the development, validation, and forensic casework application of a liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method capable of definitively quantifying ∆8-THC, ∆9-THC, ∆10-THC, their carboxy-metabolites, and cannabidiol (CBD) in urine. Method performance was compared to a prior ∆9-cTHC confirmation protocol, and prevalence of isomers was assessed in 220 anonymous forensic samples.

Methodology and Instrumentation

Sample preparation involved beta-glucuronidase hydrolysis, protein precipitation with formic-acidified methanol, and addition of deuterated internal standards. Chromatographic separation was achieved on a Waters CORTECS C18+ solid-core column (1.6 µm, 2.1×50 mm) using a 5.5 min gradient from 55% to 95% acetonitrile with 0.1% formic acid. Detection employed a Xevo TQD tandem mass spectrometer in positive electrospray mode with targeted multiple reaction monitoring for each isomer. Ultrapure reagents and certified reference materials ensured traceability and accuracy.

• Instrument: Waters ACQUITY UPLC I-Class FTN coupled to Xevo TQD MS
• Column: Waters CORTECS UPLC C18+ (1.6 µm, 2.1×50 mm)
• Mobile phases: 0.1% formic acid in water and acetonitrile
• Run time: 5.5 min per injection
• Internal standards: ∆9-THC-D3, ∆9-cTHC-D3, CBD-D3

Main Results and Discussion

The optimized method achieved baseline resolution of isobaric analytes within 5.5 min. Matrix effect evaluation in twelve urine sources revealed mild ion suppression for metabolites (up to –28%) and enhancement for parent THC isomers (up to +32%), effectively normalized by isotope-labeled standards. Precision across quality controls (%CV) ranged from 4% to 17% and accuracy bias remained under 15%. Limit of detection was 4 ng/mL. Carryover was below 0.03%. Comparison with the legacy ∆9-cTHC method demonstrated elimination of ∆8-cTHC interference in ∆9-cTHC quantitation. Casework analysis showed ∆9-cTHC prevalence at 99%, ∆8-cTHC at 14%, and CBD at 10%, with co-excretion patterns indicating concurrent use.

Benefits and Practical Applications

• Unambiguous identification of multiple THC isomers and their metabolites
• High throughput suitability for forensic laboratory workflows
• Mitigation of false positives and improved legal defensibility
• Adaptable platform for emerging cannabinoid derivatives

Future Trends and Opportunities

Expanding analytical scope to novel synthetic cannabinoids will be critical as new psychoactive compounds enter the market. Exploring urinary isomer glucuronide ratios may offer insights into product composition and user exposure. Integration with high-resolution MS and automation can further enhance selectivity and throughput. Ongoing collaboration between instrumentation developers and forensic laboratories will support dynamic method adaptation.

Conclusion

The validated isomer-selective UPLC-MS/MS procedure delivers robust, rapid, and accurate confirmation of ∆8-THC, ∆9-THC, ∆10-THC, and CBD metabolites in urine. It overcomes limitations of previous methods, ensuring reliable forensic determinations and supporting evolving analytical needs in toxicology casework.