Quantitative Analysis of THC and its Metabolites in Whole Blood Using LC-MS/MS for Toxicology and Forensic Laboratories

Importance of the topic

Cannabis use and legalization have driven increased demand for reliable quantification of Δ9-tetrahydrocannabinol (THC) and its metabolites in whole blood. Accurate measurement is essential in forensic toxicology to assess recent exposure and impairment, yet the complex blood matrix and low analyte levels pose analytical challenges.

Objectives and study overview

This work presents a streamlined LC-MS/MS method to quantitate THC, 11-hydroxy-THC (THC-OH), and 11-nor-9-carboxy-THC (THC-COOH) in whole blood. The goals were to simplify sample preparation, enhance extract cleanliness, and achieve high sensitivity and reproducibility.

Methodology and instrumentation used

Samples were subjected to cell lysis and protein precipitation (zinc sulfate/ammonium acetate followed by formic acid in acetonitrile), then directly loaded onto Oasis PRiME HLB µElution plates without conditioning or equilibration. Analytes were eluted in 90:10 acetonitrile:isopropanol for direct injection.

Used Instrumentation:

• Oasis PRiME HLB µElution Plate
• ACQUITY UPLC I-Class with BEH C18 column (1.7 µm, 2.1×50 mm)
• Xevo TQ-S mass spectrometer

Chromatography: 4 min gradient at 0.6 mL/min. MS: positive-mode ESI with optimized MRM transitions and deuterated internal standards.

Main results and discussion

Recovery for all analytes exceeded 85% (RSD 5–7%), and matrix effects were under 15%. Phospholipid removal surpassed 99% compared to protein precipitation, reducing potential interferences. All compounds eluted within 3 min with symmetrical peak shapes. Calibration curves showed excellent linearity (R² > 0.998) over 0.05–100 ng/mL. Lower limits of quantification were 0.05 ng/mL for THC and THC-COOH, and 0.1 ng/mL for THC-OH. Quality control samples were within ±10% accuracy and 2–4% precision.

Benefits and practical applications of the method

The protocol eliminates SPE conditioning steps, reduces solvent consumption, and enables direct injection, streamlining laboratory workflow. High sensitivity meets forensic cut-off concentrations, while cleaner extracts extend column life and minimize MS source maintenance.

Future trends and potential applications

Adapting this approach to high-throughput and HPLC platforms will broaden accessibility. Expansion to include panels of additional drugs and metabolites can support comprehensive toxicological screening. Clinical therapeutic monitoring and doping control are further potential applications.

Conclusion

The Oasis PRiME HLB µElution LC-MS/MS method provides a rapid, robust, and reproducible solution for quantifying THC and its metabolites in whole blood, addressing key forensic toxicology requirements.

References

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2. Bansal S, DeStefano A: Key elements of bioanalytical method validation for small molecules. The AAPS Journal 9(1):E109–E114 (2007).
3. Lee R, Saussereau E, Lacroix C, Wood M: Quantitative Analysis of Cannabinoids in Whole Blood using UPLC-MS/MS for Forensic Laboratories. Waters Application Note 720004700EN (2013).