SOHT: ANALYSIS OF CARBOXY-THC USING UPLC-MS/MS

Significance of the Topic

Hair analysis provides a non-invasive and easily stored biological matrix offering extended detection windows for drug use. Incorporation mechanisms allow substances to bind into the hair shaft as it grows at approximately 1 cm per month, enabling retrospective monitoring of drug exposure over several months. Confirming Δ9-tetrahydrocannabinol metabolite levels in hair, specifically 11-nor-9-carboxy-THC (cTHC), is essential to distinguish active cannabis use from passive environmental exposure.

Objectives and Overview of the Study

This work aimed to develop and validate a sensitive and robust UPLC-MS/MS method for quantifying cTHC in human hair in compliance with Society of Hair Testing (SoHT) confirmation cut-off guidelines (0.2 pg/mg). The study outlines sample preparation, chromatographic separation, mass spectrometric detection, and method performance evaluation.

Methodology

25 mg hair samples were incubated with M3 reagent at 100 °C for 60 minutes, spiked with deuterated internal standard (cTHC-d3), and subjected to solid-phase extraction on an Oasis PRiME HLB cartridge. Following washes with acetonitrile and hexane, analytes were eluted with acetonitrile/methanol, evaporated, and reconstituted in 50% methanol with ammonia.

Instrumentation Used

UPLC separation was performed on a Waters ACQUITY UPLC I-Class FTN system equipped with a 2.1 × 150 mm BEH C18 column (1.7 µm) at 55 °C. Mobile phases comprised 0.4 mM ammonium fluoride with 0.0025% ammonium hydroxide (A) and methanol (B) under a gradient elution at 0.35 mL/min. Detection employed a Xevo TQ-Absolute triple quadrupole MS in negative ESI mode, monitoring MRM transitions for cTHC (m/z 434.1 → 191.0, 245.1) and cTHC-d3 (m/z 346.1 → 248.1).

Main Results and Discussion

The assay demonstrated linear calibration from 0.2 to 10 pg/mg with excellent correlation and residuals. Signal-to-noise ratios at the lower limit of quantification (0.2 pg/mg) exceeded acceptance criteria for both quantifier and qualifier ions. Precision and accuracy were confirmed across mixed hair samples (n=5) with triplicate injections, showing minimal variability. Carryover tests using a high-level standard (5 pg/mg) followed by blank injections revealed no detectable contamination.

Benefits and Practical Applications of the Method

• Meets SoHT confirmation cut-offs for forensic hair testing of cTHC.
• Enables non-invasive, supervised collection and long-term exposure assessment.
• Delivers sub-pg/mg sensitivity with high throughput and low carryover.

Future Trends and Potential Applications

Advances in microextraction techniques and automation may streamline sample preparation. High-resolution mass spectrometry could expand metabolite panels and improve specificity. Integration with bioinformatics and machine learning will enhance data interpretation and forensic intelligence.

Conclusion

The validated UPLC-MS/MS workflow provides a rapid, accurate, and sensitive approach for detecting cTHC in hair at sub-pg/mg levels, fulfilling SoHT requirements. Its robustness and minimal carryover support reliable forensic toxicology applications for retrospective cannabis use monitoring.

References

1. Cooper GAA, Kronstrand R, Kintz P. Society of Hair Testing guidelines for drug testing in hair. Forensic Science International. 281;2012:20-24.