Analysis of THC Metabolites in Urine by GC-MS/MS

Importance of the Topic

Cannabis remains one of the most commonly used psychoactive substances worldwide. Quantifying its main active component Δ9-THC and related metabolites in urine is essential for forensic toxicology, clinical monitoring, workplace drug testing, and anti-doping programs. Reliable detection at trace levels helps distinguish recent use, assess impairment, and ensure compliance with evolving legal and regulatory frameworks.

Objectives and Study Overview

This study, conducted by Shimadzu Corporation and Showa University, aimed to establish a robust GC-MS/MS method for simultaneous analysis of Δ8-THC, Δ9-THC, and their primary hydroxy- and carboxy-metabolites in urine. Four sample pretreatment strategies were compared to identify the most effective approach for trace-level quantitation.

Methodology and Instrumentation

• Instrumentation Used:
  • Shimadzu GCMS-TQ8040 NX triple quadrupole GC-MS/MS
  • AOC-30i autosampler
  • SH-I-5Sil MS capillary column (30 m × 0.25 mm I.D., 0.25 µm film)
• GC Conditions: Injection temp. 280 °C (splitless), oven ramp 150 °C (1 min) → 320 °C at 15 °C/min (3 min), He at 1.44 mL/min
• MS Conditions: Ion source 230 °C, interface 280 °C, MRM acquisition with optimized precursor-product transitions
• Sample Pretreatment Workflow:
  1. Hydrolysis (alkaline or enzymatic) to release glucuronide conjugates
  2. Extraction comparison: ISOLUTE SLE+, MonoSpin C18-CX, Shimadzu micro QuEChERS, liquid-liquid (hexane/ethyl acetate)
  3. Drying under nitrogen, reconstitution in acetonitrile, TMS derivatization with BSTFA + 1% TMCS at 70 °C for 30 min

Key Results and Discussion

• Liquid-liquid extraction achieved the highest recoveries (71–81%) across all analytes, despite 20–30% loss from hydrolysis.
• MRM detection delivered a lower limit of quantitation of 1 ng/mL for each THC metabolite, with S/N ratios exceeding 200.
• Calibration curves were linear over 1–500 ng/mL with correlation coefficients ≥ 0.997.
• Intra-day accuracy ranged 97–106% and precision (%RSD) was ≤ 3.2% at low (15 ng/mL) and high (250 ng/mL) concentrations.

Benefits and Practical Applications

• The two-stage mass filtering of MRM minimizes matrix interferences, enabling reliable trace-level quantitation in complex urine samples.
• The optimized protocol is well suited for high-throughput forensic and clinical laboratories conducting routine cannabinoid screening.

Future Trends and Opportunities

• Automation of sample pretreatment to enhance throughput and reproducibility.
• Use of high-resolution or time-of-flight MS systems to broaden analyte coverage and improve specificity.
• Standardization of cannabinoid testing guidelines in response to shifting legal frameworks worldwide.

Conclusion

The developed GC-MS/MS method, incorporating liquid-liquid extraction and MRM detection, offers sensitive (1 ng/mL), accurate, and precise quantitation of Δ8- and Δ9-THC metabolites in urine. This approach meets the demands of forensic, clinical, and anti-doping testing environments.

References

• II-5 Cannabis Test Methods, Ed. by The Pharmaceutical Society of Japan: Toxicological Test Methods and Annotations; 2017.