Quantitative analysis of cannabinoids in urine and oral fluid using reactive paper spray tandem mass spectrometry for clinical research and forensic toxicology

Significance of the Topic

Cannabinoid testing in biological fluids is critical for workplace safety, clinical trials and forensic investigations. Conventional confirmatory assays rely on GC-MS or LC-MS with extensive sample preparation and long analysis times, creating backlogs and limiting throughput. Rapid, sensitive and cost-effective methods that meet regulatory cutoff levels for THC in oral fluid and its metabolite THC-COOH in urine can improve laboratory efficiency and support timely decision-making.

Objectives and Study Overview

The study aims to develop a high-throughput, mass spectrometry-based workflow for quantitative determination of THC in oral fluid and THC-COOH in urine. The method employs reactive paper spray tandem mass spectrometry with on-paper derivatization to achieve sensitivity that meets confirmatory cutoff values using a TSQ Fortis Plus triple quadrupole instrument and a VeriSpray PaperSpray ion source.

Instrumental Setup

The analytical system consists of:

• TSQ Fortis Plus triple quadrupole mass spectrometer
• VeriSpray PaperSpray ion source with automated plate loader holding up to ten sample plates (240 samples unattended)
• Thermo Scientific TraceFinder software for data acquisition and processing

Methodology and Analytical Procedure

Sample preparation uses on-paper derivatization with Fast Red RC diazonium salt deposited and dried on paper strips. Oral fluid or urine is spiked with deuterated internal standards, diluted in methanol solution (70:30), and applied onto the derivatized spots. Reactive paper spray is initiated by adding dichloromethane solvent aliquots followed by electrospray ionization at +3.8 kV. Selected reaction monitoring transitions are optimized for derivatized THC and THC-COOH. Calibration curves cover 0.5 to 250 ng/mL with 1/x weighting. Method validation follows SWGTOX guidelines, evaluating accuracy, repeatability and reproducibility over five days.

Key Results and Discussion

Calibration for THC in oral fluid and THC-COOH in urine demonstrated excellent linearity (R2 ≥ 0.99). Lower limits of quantitation were 4 ng/mL for THC and 10 ng/mL for THC-COOH, satisfying regulatory cutoffs. Quality control samples at low (9 ng/mL), medium (80 ng/mL) and high (200 ng/mL) levels showed bias within ±15% and precision below 15% both within and between runs. Total analysis time per sample, including solvent addition and spray, was approximately 2.2 minutes, greatly improving throughput compared to traditional chromatography-based workflows.

Benefits and Practical Applications

• Rapid analysis reduces per-sample cost and increases laboratory throughput
• Minimal sample preparation and on-paper derivatization simplify workflow
• Sensitivity and specificity meet forensic and clinical confirmatory testing requirements
• Non-invasive sample collection for oral fluid facilitates field applications

Future Trends and Opportunities

Advances in reactive paper spray chemistries may extend to other drug classes and metabolites. Integration with automated sample handling platforms can further minimize hands-on time. Coupling with high-resolution mass analyzers and multiplexed reaction monitoring could enhance selectivity and coverage. As regulatory agencies accept non-chromatographic MS approaches, PS-MS methods may become standard for rapid toxicology screening and confirmation.

Conclusion

Reactive paper spray tandem mass spectrometry with on-paper derivatization provides a fast, sensitive and robust alternative to conventional chromatographic methods for quantitative cannabinoid analysis in urine and oral fluid. The workflow meets forensic and clinical cutoff requirements, reduces analysis time to under 3 minutes per sample and supports high throughput in toxicology laboratories.

Reference

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