SOFT: Analysis of carboxy-THC in hair using UPLC-MS/MS

Importance of the Topic

The metabolism of Δ9-tetrahydrocannabinol produces 11-nor-9-carboxy-THC (carboxy-THC), a key biomarker for cannabis exposure. Hair analysis offers a noninvasive, stable matrix to monitor prolonged drug use, meeting forensic and clinical needs under Society of Hair Testing guidelines.

Goals and Study Overview

This work aims to establish a reliable UPLC-MS/MS assay for quantifying carboxy-THC in hair at sub-pg/mg levels, validating sensitivity, specificity, and robustness in accordance with SoHT confirmation cut-offs.

Methodology and Instrumentation

Sample Preparation:

• Hair decontamination, scissor-mincing into 1–2 mm segments.
• Weighing 25 mg samples and spiking with carboxy-THC calibrators (0.2–10 pg/mg) and carboxy-THC-D3 internal standard.
• Incubation with M3 buffer at 100 °C for 60 min.
• SPE cleanup using OASIS PRiME HLB cartridges with acetonitrile and hexane washes; elution in acetonitrile/methanol (9:1), solvent evaporation, reconstitution in 50% methanol/ammonia.

UPLC-MS/MS Conditions:

• System: Waters ACQUITY UPLC I-Class PLUS with Xevo TQ-Absolute.
• Column: ACQUITY Premier BEH C18, 1.7 μm, 2.1×150 mm at 55 °C.
• Mobile phases: 0.4 mM NH4F/0.0025% NH4OH in water (A), methanol (B); flow 0.35 mL/min; gradient from 70/30 to 2/98 over 4.2 min.
• Injection: 15 μL; sample at 10 °C.
• MS: ESI negative mode; MRM transitions m/z 343.1>191.0 (quantifier), 343.1>245.1 (qualifier), and 346.1>248.1 for internal standard; cone voltage 30 V; collision energy 31–33 eV.

Main Results and Discussion

• Linearity: 0.2–10 pg/mg, R²>0.99; accuracy within ±15% (±20% at LLOQ).
• LLOQ: 0.2 pg/mg with S/N ~18.9 (quantifier) and ~15.3 (qualifier).
• Robustness: intra- and inter-batch %RSD ≤8.4%; average within-sample %RSD 1.8% for mixed hair samples.
• Matrix effects: 36% ion suppression if uncorrected; fully compensated by isotopically labeled internal standard (matrix factor 100%).
• No detectable carryover after a 5 pg/mg injection.

Benefits and Practical Applications

The validated method enables noninvasive, sensitive detection of carboxy-THC in hair for forensic toxicology, supporting long-term exposure monitoring, casework confirmation, and compliance with international guidelines.

Future Trends and Opportunities

Advances may include integration of high-resolution mass spectrometry for broader cannabinoid profiling, microextraction techniques to reduce solvent use, automation for high-throughput screening, and exploration of additional biomarkers to distinguish passive exposure.

Conclusion

This UPLC-MS/MS workflow demonstrates the capability to quantify carboxy-THC at sub-pg/mg levels in hair with high accuracy, precision, and robustness, fulfilling SoHT confirmation criteria and enhancing forensic practice.

References

1. Cooper G.A.A., Kronstrand R., Kintz P. Society of Hair Testing guidelines for drug testing in hair. Forensic Sci Int. 281 (2012):20–24.
2. Society of Hair Testing. Statements concerning the examination of drugs in human hair. (2023).