Analysis of THC and an Extended Metabolite Suite from Oral Fluid Using ISOLUTE® SLE+ Supported Liquid Extraction Columns Prior to LC-MS/MS

Significance of the Topic

The analysis of delta-9-tetrahydrocannabinol (THC) and its related metabolites in oral fluid is critical for forensic toxicology, workplace testing and clinical monitoring. Oral fluid sampling offers a noninvasive matrix with close correlation to blood levels, facilitating timely detection of recent cannabis use and metabolic profiling.

Objectives and Study Overview

This work aimed to develop and validate a robust, high-throughput method for extraction and quantitation of THC, its acidic form THCA-A, major cannabinoids and an extended suite of metabolites from oral fluid collected using Quantisal devices. Key goals included maximizing analyte recovery, minimizing matrix effects and achieving low limits of quantitation using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Methodology and Instrumentation

• Sample Preparation
  • Supported Liquid Extraction using ISOLUTE SLE+ columns (400 µL or 1 mL capacity).
  • Oral fluid samples were acidified to pH 3.6 with 10 µL concentrated formic acid and vortexed.
  • Sequential elution: two aliquots of methyl tert-butyl ether (MTBE) followed by one aliquot of hexane.
  • Evaporative drying under nitrogen (TurboVap LV) and reconstitution in 0.1% formic acid in H₂O/ACN (60/40, v/v).
  
  
• Chromatography and Detection
  • UPLC: Waters ACQUITY system with Phenomenex Kinetex XB-C18 column (50×2.1 mm, 2.6 µm).
  • Mobile phase: Gradient of 0.1% formic acid in water (A) and in acetonitrile (B) over 4 min. Flow rate 0.35 mL/min.
  • MS/MS: Waters Ultima Pt with ESI+ and multiple reaction monitoring (MRM).
  • Optimized temperatures: desolvation 350 °C, source 100 °C.

Main Results and Discussion

• Recoveries exceeded 64% across all analytes with relative standard deviations (RSDs) below 10% for both 300 µL and 800 µL load volumes.
• Calibration curves (1–200 ng/mL) showed excellent linearity (r² ≥ 0.994). Limits of quantitation as low as 1–2 ng/mL were achieved with a 5 µL injection.
• Acidic pH pre-treatment (pH 3.6) was essential to recover glucuronide conjugates, particularly THC-COOH-glucuronide, which was not extracted at higher pH.

Benefits and Practical Application

This method combines the cleanliness and automation potential of SLE with rapid UPLC-MS/MS analysis, offering:

• High throughput suitable for routine forensic and clinical laboratories.
• Reliable quantitation of parent cannabinoids and metabolites for comprehensive metabolic profiling.
• Compatibility with common oral fluid collection devices, reducing sample variability.

Future Trends and Possibilities

Anticipated developments include integration with fully automated sample preparation platforms, further miniaturization of LC columns for even faster analysis and expansion to additional phase II metabolites. Advances in high-resolution MS could enable broader screening of novel synthetic cannabinoids in oral fluid.

Conclusion

The described ISOLUTE SLE+ supported liquid extraction combined with UPLC-MS/MS offers a validated, efficient workflow for sensitive, reproducible analysis of THC and its metabolites in oral fluid. This approach addresses key challenges in forensic and clinical testing by delivering high recoveries, low limits and streamlined throughput.

Reference

• Biotage Application Note AN815 (2014) Analysis of THC and an Extended Metabolite Suite from Oral Fluid Using ISOLUTE SLE+ Supported Liquid Extraction Prior to LC-MS/MS.