Quantitative Separation of THC Isomers and Metabolites from Whole Blood

Importance of the topic

The accurate separation and quantification of psychoactive THC isomers (Δ8-, Δ9-, and Δ10-THC) and their metabolites in whole blood is critical for forensic toxicology, particularly in driving under the influence investigations. Isobaric interferences from nonpsychoactive cannabinoids such as CBD and exo-THC complicate conventional mass spectrometric analyses, potentially leading to false positives or misquantitation. A robust analytical method that minimizes matrix effects and reliably distinguishes psychoactive compounds supports legal and clinical assessments.

Aims and Study Overview

This application note presents a rapid 15-minute LC/MS/MS workflow combining Agilent Captiva Enhanced Matrix Removal–Lipid cartridges with an InfinityLab Poroshell 120 PFP column on Agilent triple quadrupole instruments. The goal was to achieve baseline chromatographic separation and sensitive quantification of Δ8-, Δ9-, Δ10-THC isomers and their hydroxy and carboxy metabolites in whole blood extracts.

Methodology and Instrumentation

Sample preparation employs:

• 500 µL of whole blood loaded onto a 3 mL Agilent Captiva EMR–Lipid cartridge.
• Protein precipitation with 1.2 mL cold acetonitrile/methanol (85:15, v/v), followed by a 5 minute equilibration and positive‐pressure elution.
• Evaporation of the eluent under nitrogen and reconstitution in 100 µL of 50:50 methanol/water.

Chromatographic and MS conditions include:

• Agilent InfinityLab Poroshell 120 PFP column (2.1 × 100 mm, 2.7 µm) at 55 °C with a 13.5 minute gradient (water + 0.1% formic acid/methanol).
• Injection volume 5 µL, flow rate 0.5 mL/min.
• Detection on Agilent 6475 or Ultivo triple quadrupole MS with Jet Stream ESI in positive ion mode, operating dynamic MRM.

Main Results and Discussion

The method produced baseline separation of all target THC isomers and metabolites from isobaric compounds within a single 15 minute run. Sensitivity reached low ng/mL levels, with quantitative ranges of 5–400 ng/mL in blood and correlation coefficients above 0.998 for all analytes. Recovery experiments demonstrated 80–120% analyte extraction efficiency using EMR cartridges. Chromatograms illustrated clear resolution of psychoactive THC isomers from CBD and exo-THC, confirming the method’s specificity.

Benefits and Practical Applications

This streamlined workflow offers:

• High throughput suitable for forensic casework.
• Reduced matrix interference and minimal sample handling time.
• Reliable differentiation of psychoactive THC isomers, supporting legal and clinical toxicology decisions.

Future Trends and Potential Applications

Emerging directions include:

• Automation of the sample preparation to further increase throughput.
• Extension to alternative matrices such as oral fluid or urine for broader toxicology screening.
• Integration with high‐resolution mass spectrometry to characterize novel cannabinoid analogs.

Conclusion

The combined use of Agilent Captiva EMR–Lipid cartridges with LC/MS/MS provides a rapid, sensitive, and selective platform for separating and quantifying Δ8-, Δ9-, and Δ10-THC isomers and their metabolites in whole blood. Its robust performance across critical analytical metrics makes it a valuable tool for forensic toxicology laboratories.

Reference

Stevens J; Zhao L. Efficient Quantitative Analysis of THC and its Metabolites in Whole Blood Using Agilent Captiva EMR–Lipid and LC-MS/MS. Agilent Technologies Application Note 5991-8635EN, 2020.