Extraction of THC and Metabolites Including 11-nor-9-carboxy-Δ9-THC Glucuronide from Urine Using ISOLUTE® SLE+ Prior to LC-MS/MS Analysis

Importance of the Topic

Accurate measurement of Δ9-tetrahydrocannabinol (THC) and its metabolites in urine is critical for monitoring substance use and compliance assessment in clinical, forensic, and workplace settings.

Study Objectives and Overview

This application note presents optimized protocols using ISOLUTE SLE+ supported liquid extraction in both 200 µL plate and 1 mL column formats to simultaneously extract THC, 11-OH-THC, THC-COOH, THC-COOH glucuronide, cannabidiol, and cannabinol from urine samples prior to LC-MS/MS analysis. The work aims to preserve glucuronide integrity by controlling pH during sample pre-treatment and to achieve high recovery and reproducibility.

Methodology

Sample Pre-treatment:

• Urine samples were diluted 1:1 (v/v) with 25 mM dibutylammonium acetate buffer to stabilize glucuronides and prevent hydrolysis.
• Vortex mixing ensured homogeneous sample-buffer mixtures.

Extraction Protocols:

• 200 µL SLE+ plate format (part no. 820-0200-P01): Load 200 µL diluted urine, allow adsorption for 5 minutes, extract with 1 mL ethyl acetate under gravity followed by vacuum.
• 1 mL SLE+ column format (part no. 820-0140-C): Load 1 mL diluted urine, adsorb for 5 minutes, perform two 2.5 mL ethyl acetate extractions under gravity, then apply pressure.

Post-elution and Reconstitution:

• Evaporate eluents to dryness at 40°C using SPE Dry or TurboVap.
• Reconstitute in 200 µL of 0.1% formic acid in water/acetonitrile (70:30, v/v).

Instrumentation

• ISOLUTE SLE+ supported liquid extraction plate and 1 mL column (Biotage).
• Waters ACQUITY UPLC system with BEH C18 column (1.7 µm, 100 × 2.1 mm).
• Premier XE triple quadrupole mass spectrometer with electrospray ionization.

Main Results and Discussion

The method achieved analyte recoveries above 85% for both 200 µL and 500 µL urine sample volumes spiked at 40 ng/mL, with relative standard deviations below 10%. MRM chromatograms demonstrated clear resolution of all target compounds. The optimized pH conditions preserved the glucuronide metabolite, while the SLE+ format eliminated emulsions and reduced handling time compared to traditional liquid-liquid extraction.

Benefits and Practical Applications

• High throughput sample preparation suitable for clinical and forensic laboratories.
• Enhanced analyte recovery and reproducibility without emulsion issues.
• Compatibility with standard LC-MS/MS workflows for quantitative analysis.

Future Trends and Opportunities

Potential developments include the automation of SLE+ workflows, integration with high-resolution mass spectrometry for broader metabolite profiling, adaptation to other glucuronidated drug classes, and miniaturized formats to reduce solvent consumption and improve sustainability.

Conclusion

The ISOLUTE SLE+ extraction protocols provide a streamlined, reliable, and high-recovery method for simultaneous extraction of THC and its key metabolites from urine prior to LC-MS/MS analysis, addressing pH sensitivity and throughput requirements in bioanalytical laboratories.

References

1. The data in this application note was originally presented in poster form at the 2011 combined TIAFT/SOFT annual conference.
2. Scheidweiler KB, Desrosiers NA, Huestis MA. Clin Chim Acta. 2012 Nov 20;413(23-24):1839–1847. doi:10.1016/j.cca.2012.06.034.