Quantitative Determination of Urinary COOH-THC for Forensic Toxicology: Comparison to a Validated Reference Method

Importance of the Topic

The quantification of 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (COOH‐THC) in urine is critical for forensic toxicology, workplace drug testing, and clinical monitoring. Reliable methods that minimize sample handling and reduce analyte loss are in high demand, especially as cannabis regulations evolve.

Objectives and Study Overview

This study presents an interlaboratory comparison between a simplified solid‐phase extraction (SPE) workflow using Oasis PRiME HLB µElution Plates and a fully validated reference method from an external laboratory. Twenty‐five authentic urine samples spanning 6.70–458 ng/mL COOH‐THC were analyzed to assess accuracy, precision, and agreement.

Methodology

Sample preparation involved enzymatic hydrolysis of COOH‐THC glucuronide by adding KOH and heating at 65 °C for 2 hours. After internal standard addition, 75 µL of hydrolyzed urine was loaded directly onto Oasis PRiME HLB µElution Plate wells. Samples were washed with 25:75 methanol:water and eluted with 60:40 acetonitrile:isopropanol. Eluates were diluted and 5 µL injected.

Used Instrumentation

• Oasis PRiME HLB µElution Plate (p/n 186008052)
• ACQUITY UPLC I-Class System
• ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 × 50 mm, p/n 186002350)
• Xevo TQ-S Triple Quadrupole Mass Spectrometer

Results and Discussion

Deming regression yielded a slope of 0.995 and correlation coefficient (R) of 0.998, indicating excellent agreement between methods. Bland-Altman analysis at 95% limits of agreement confirmed consistency, with 78% of sample pairs within ±20% bias (FDA-GLP requires ≥67%). A slight negative bias in authentic samples, absent in surrogate matrix standards, suggests minor matrix suppression differences between methods. Overall, simplified SPE without conditioning or evaporation matched the performance of the validated protocol.

Benefits and Practical Applications

• Streamlined sample preparation by eliminating conditioning, evaporation, and reconstitution steps
• Direct concentration on SPE device reduces analyte loss and hands-on time
• High recovery and minimal matrix effects improve accuracy across a broad calibration range (5–500 ng/mL)
• Rapid turnaround supports high-throughput forensic and clinical laboratories

Future Trends and Opportunities

Advances may include full automation of µElution workflows, integration with online SPE-UPLC systems, and expansion to additional metabolites or biological matrices. Further miniaturization and coupling with high-resolution mass spectrometry could enhance sensitivity and selectivity for low‐level detection in complex samples.

Conclusion

The Oasis PRiME HLB µElution Plate method provides a fast, clean, and robust approach for COOH-THC quantification in urine. It delivers results equivalent to a validated reference method while reducing sample prep time and potential analyte loss, making it a valuable tool for forensic toxicology laboratories.

References

1. Zhang X., Danaceau J.P., Chambers E. Quantitative Analysis of THC and Metabolites in Urine with a Simple, Fast, and Clean Oasis PRiME HLB µElution Plate. Waters Application Note 720005556EN (2015).
2. Booth B., Arnold M.E., DeSilva B. Workshop Report: Crystal City V – Quantitative Bioanalytical Method Validation and Implementation: The 2013 Revised FDA Guidance. The AAPS Journal 17(2), 277–288 (2015).