Extraction of Tetrahydrocannabinol (THC) and Metabolites from Whole Blood Using ISOLUTE® SLE+ Prior to LC-MS/MS

Importance of the Topic

Toxicological screening of tetrahydrocannabinol (THC) and its metabolites in whole blood is crucial for forensic and clinical analysis. Whole blood presents challenges such as high viscosity and complex matrix components that can lead to ion suppression in LC-MS/MS. Developing a reliable sample preparation method ensures accurate detection and quantitation of analytes in postmortem and ante-mortem investigations.

Objectives and Overview of the Study

The primary goal was to evaluate a supported liquid extraction (SLE+) approach using a 96-well format to isolate THC and key metabolites from spiked whole blood. The study compared SLE+ to traditional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) workflows to determine efficiency, recovery, and matrix effect minimization.

Methodology

• Sample Pre-treatment: Add spiked whole blood (200 μL) to each SLE+ well, acidify with 0.1% formic acid (100 μL) and internal standards.
• Sample Loading: Load up to 330 μL total volume onto 400 μL SLE+ wells under slight vacuum or positive pressure, allow 5 minute absorption.
• Elution: Perform three elutions with 600 μL methyl tert-butyl ether (MTBE), facilitating flow at ~1 mL/min.
• Post-extraction: Evaporate eluent, reconstitute in 50:50 water:methanol (500 μL) for LC-MS/MS analysis.
• Analytes: Δ9-THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC and corresponding deuterated standards.

Used Instrumentation

• 96-well ISOLUTE SLE+ Supported Liquid Extraction Plate.
• Vacuum/Positive Pressure Manifolds (VacMaster-96, PRESSURE+ 96).
• HPLC: Agilent 1200 system with Phenomenex Gemini C18 column (150 × 4.6 mm, 5 μm).
• Mass Spectrometer: AB Sciex 4000 Q-Trap with Turbo Ionspray ESI source.

Main Results and Discussion

Extraction recoveries for THC and metabolites ranged from 60% to 107% across 12.5–50 ng/mL, with inter-day precision below 10% RSD over seven replicates. Matrix-induced ion suppression remained under 10%, confirming the method’s robustness. Extracted ion chromatograms demonstrated clear separation and sensitivity at 25 ng/mL levels.

Benefits and Practical Applications

• SLE+ provides rapid, reproducible cleanup with minimal solvent usage compared to LLE and SPE.
• High throughput 96-well format streamlines sample preparation for forensic and clinical laboratories.
• Reduced matrix effects improve quantitation accuracy in LC-MS/MS workflows.

Future Trends and Potential Applications

The SLE+ approach can be extended to other biologically relevant analyte classes in diverse matrices. Integration with automated liquid handling systems and novel sorbent chemistries may further enhance throughput and selectivity in routine toxicology and therapeutic drug monitoring.

Conclusion

Supported liquid extraction using ISOLUTE SLE+ in a 96-well format offers an efficient, robust method for isolating THC and metabolites from whole blood. The protocol delivers high recoveries, low matrix interference, and compatibility with LC-MS/MS analysis, making it suitable for high-throughput toxicological screening.