Automated Extraction, Derivatization and GC/MS Determination of Tetrahydrocannabinol and Metabolites in Whole Blood Using Disposable Pipette Extraction

Significance of the Topic

Accurate and efficient determination of tetrahydrocannabinol (THC) and its metabolites in whole blood is critical in forensic toxicology, requiring low detection limits and robust extraction from complex matrices. Disposable Pipette Extraction (DPX) offers rapid, reproducible solid-phase extraction with minimal solvent use, and when combined with automated systems can greatly improve throughput and consistency.

Objectives and Study Overview

• Develop a fully automated DPX-based method to extract THC, hydroxy-THC (OH-THC) and carboxy-THC (COOH-THC) from whole blood
• Integrate sample introduction, concentration and derivatization directly in the GC inlet using a GERSTEL MultiPurpose Sampler (MPS) and Cooled Injection System (CIS)
• Achieve detection limits of at least 1 ng/mL for THC and OH-THC and 2 ng/mL for COOH-THC with high precision

Methodology and Instrumentation

Samples (0.5 mL) were spiked with deuterated internal standards and protein precipitated with acetonitrile. After centrifugation and acidification, automated extraction was performed with 1 mL DPX-RP tips on a dual-rail GERSTEL MPS 2, using three 0.7 mL extractions, a 0.5 mL water wash and elution with dichloromethane. Eluates were delivered to a GERSTEL CIS 4 PTV inlet for large volume injection, where solvent venting, analyte concentration and derivatization (1:1 BSTFA:MTBSTFA) occurred in a single automated “sandwich” injection. The system comprised an Agilent 6890N GC with 5975 MSD, GERSTEL CIS and MPS 2 PrepStation with DPX option.

Key Results and Discussion

• Post-precipitation recoveries were ~80 % for THC; lower apparent values reflect losses during protein removal rather than DPX inefficiency
• Limits of detection were 0.5 ng/mL for THC and OH-THC, and 2 ng/mL for COOH-THC, with coefficients of variation below 5 %
• Combined use of BSTFA and MTBSTFA ensured reproducible derivatization of all analytes, overcoming individual reagent limitations
• Automated DPX integrated with the CIS inlet completed sample preparation in under 6 minutes, enabling just-in-time processing and full utilization of GC runtime

Benefits and Practical Applications

• Fully automated DPX reduces manual handling, enhances reproducibility and minimizes solvent consumption
• Integration of extraction, concentration and derivatization streamlines workflows in forensic and clinical laboratories
• High throughput automation supports large sample batches with minimal downtime, ideal for routine THC monitoring and compliance testing

Future Trends and Potential Applications

• Optimization of sorbent materials and elution solvents to further improve recoveries of acidic metabolites
• Extension of automated DPX methods to other drug classes and biomarkers in complex matrices
• Coupling with high-resolution mass spectrometry for broad-spectrum screening of novel psychoactive substances

Conclusion

The automated DPX method using a GERSTEL MPS 2 coupled to a CIS inlet provides a fast, reliable and sensitive approach for simultaneous GC/MS analysis of THC and its metabolites in whole blood. This workflow meets strict forensic requirements while offering significant improvements in throughput and reproducibility.

References

• Schroeder JL, Marinetti LJ, Smith RK, Brewer WE, Clelland BL, Morgan SL. The analysis of Δ9-tetrahydrocannabinol and metabolites in whole blood and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine using disposable pipette extraction with confirmation and quantification by gas chromatography-mass spectrometry. Journal of Analytical Toxicology, in press.