MRM and Product Ion Scan Analysis of THC Metabolites in Urine Using LC-MS/MS

Importance of the Topic

Cannabis regulation is evolving worldwide, with both legalization and stricter enforcement implemented in different jurisdictions. Monitoring Δ9-tetrahydrocannabinol (Δ9-THC) and its primary metabolites in biological fluids is vital for forensic toxicology, clinical toxicology, and workplace drug testing. Sensitive and reliable analytical methods enable definitive proof of impairment or compliance in legal and medical settings.

Objectives and Overview

This study aims to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method that combines multiple reaction monitoring (MRM) for quantification and product ion scanning for confirmatory identification of Δ9-THC and its metabolites (Δ9-THC-OH, Δ8-THC-OH, Δ9-THC-COOH, Δ8-THC-COOH) in urine. The approach targets sub-ng/mL detection without chemical derivatization and leverages a standardized mobile phase for compatibility with forensic screening workflows.

Methodology and Instrumentation

The procedure involves:

• Sample pretreatment by liquid–liquid extraction: urine (200 µL) is acidified (pH <2.5), spiked with internal standards, extracted twice with hexane:ethyl acetate (7:1), hydrolyzed under alkaline conditions (10 N NaOH, 50 °C, 15 min), re-extracted, and dried under nitrogen.
• Reconstitution in water:methanol (1:1, 100 µL) for analysis.

Instrumentation Used:

• UHPLC: Nexera XS system.
• Column: Raptor ARC-18 (100 mm×3.0 mm I.D., 1.8 µm).
• Mobile phase A: 10 mM ammonium formate + 0.1 % formic acid in water; B: same additive in methanol. Gradient: 70 % B (0–9 min) → 85 % B (12–18 min) → 70 % B (18–23 min) at 0.4 mL/min.
• Mass spectrometer: LCMS-8050 triple quadrupole with ESI in positive mode. Gas: nebulizing 3 L/min, drying 5 L/min, heating 15 L/min. Interface 400 °C, DL 150 °C, heat block 400 °C.

Main Results and Discussion

The method achieved a detection limit of 1 ng/mL for all analytes without derivatization. Calibration curves from 1–500 ng/mL exhibited excellent linearity (R ≥ 0.9995). Intra-day accuracy ranged from 103 % to 110 % and precision (RSD) was below 6.6 % at both 15 ng/mL and 250 ng/mL levels. Product ion scans at 15 ng/mL provided confirmatory fragmentation patterns matching reference spectra, enhancing identification confidence.

Benefits and Practical Applications

This workflow eliminates derivatization, reduces sample preparation time, and maintains high sensitivity suitable for forensic certification. Using the same mobile phases as Shimadzu’s LC-MS/MS Forensic Toxicology Database optimizes method transfer and allows rapid column exchange to switch between targeted THC metabolite analysis and broader drug/toxin screening.

Future Trends and Potential Applications

Integration with automated sample preparation and high-resolution mass spectrometry could further improve throughput and specificity. Expanding the assay to additional cannabinoids and matrices (e.g., saliva, hair) will support comprehensive forensic and clinical toxicology. Coupling with portable MS platforms may enable on-site drug testing and real-time monitoring in roadside or workplace controls.

Conclusion

The validated LC-MS/MS method on the LCMS-8050 provides robust quantification and confirmation of Δ9-THC and its metabolites in urine at low ng/mL levels, meeting the demands of forensic and clinical toxicology. Its compatibility with existing screening packages simplifies implementation across laboratories.

Reference

• Shimadzu Corporation. Application News No. 01-00879-EN: MRM and Product Ion Scan Analysis of THC Metabolites in Urine Using LC-MS/MS. May 2025.
• Shimadzu Corporation. Application News No. 01-00860: Analysis of THC Metabolites in Urine by GC-MS/MS.