Optimized SPE for UPLC-MS/MS and GC-MS/MS Determination of THC and Metabolites in Urine and Blood

Importance of the Topic

Detection of ∆9-tetrahydrocannabinol (THC) and its major metabolites in biological fluids is essential for forensic toxicology, clinical diagnostics, workplace drug testing, and therapeutic monitoring. Accurate quantitation in urine and blood provides evidence of recent cannabis use, supports driving impairment assessments, and aids in research on pharmacokinetics and metabolism.

Objectives and Study Overview

The primary goals of the work were:

• To establish a unified solid-phase extraction (SPE) protocol applicable to both blood and urine specimens for the three analytes: THC, hydroxy-THC (OH-THC), and carboxy-THC (COOH-THC).
• To develop a rapid and sensitive UPLC-MS/MS method for direct analysis of extracted samples without derivatization.
• To implement a tandem GC-MS/MS workflow including derivatization for confirmatory analysis.

Methodology and Instrumentation

Sample Preparation:

• Blood: Protein precipitation with acetonitrile, centrifugation, dilution with aqueous ammonia, then loading onto an Oasis MAX cartridge.
• Urine: Base hydrolysis (NaOH, 60 °C), pH adjustment, acetonitrile addition, and SPE on the same cartridge type.

SPE Protocol:

• Cartridge conditioning, sample load, two sequential washes to remove polar interferences, and elution with solvent mixtures selected to maximize analyte recovery while minimizing co-extractives.
• Dry-down under vacuum; reconstitution in appropriate solvents for LC or GC analysis.

UPLC-MS/MS Conditions:

• Instrument: Waters Acquity UPLC coupled to Quattro Premier XE tandem mass spectrometer.
• Column: BEH C18 (2.1 × 100 mm); mobile phases 0.1% formic acid in water (A) and acetonitrile (B); 400 µL/min flow; 5-minute gradient 60→90% B.
• MRM transitions monitored in both positive and negative electrospray modes with deuterated internal standards.

GC-MS/MS Conditions:

• Instrument: Agilent 6890 GC with Quattro micro MS/MS.
• Column: RTX-5MS (30 m × 0.025 mm, 0.25 µm); helium carrier (1 mL/min); temperature ramp from 100 °C to 240 °C.
• Derivatization: BSTFA/TMCS (99:1) at 70 °C for 15 min; MRM transitions in EI mode.

Main Results and Discussion

Recovery and matrix effects were robust across all analytes, with 85–93% recoveries and ion suppression below 10%. Calibration curves in blood and urine showed linearity from 0 to 100 ng/mL (r²>0.99) with limits of quantitation below 5 ng/mL. Intraday and interday precision for internal standard areas remained within 10% RSD. UPLC-MS/MS provided a total run time of five minutes, enabling high throughput, while GC-MS/MS offered confirmatory specificity in a 25-minute cycle. Performance matched or exceeded that of competitor silica-based SPE cartridges.

Benefits and Practical Applications

• Single SPE workflow simplifies laboratory operations for both biological matrices and analytical platforms.
• High sensitivity and precision support low-level detection and reliable quantitation in forensic and clinical settings.
• Fast UPLC analysis accelerates turnaround time; GC-MS/MS provides orthogonal confirmation.

Future Trends and Potential Applications

Advances are expected in online SPE automation, miniaturized extraction formats, and high-resolution mass spectrometry to further increase throughput and selectivity. Green solvent protocols and lab-on-chip integration may reduce sample volumes and environmental impact. Expanding panels to include newer cannabinoids and phase II metabolites will address evolving regulatory and research needs.

Conclusion

The optimized SPE protocol coupled with UPLC-MS/MS and GC-MS/MS enables efficient, sensitive, and reproducible determination of THC, OH-THC, and COOH-THC in urine and blood. This unified workflow enhances laboratory productivity and ensures reliable data for forensic toxicology, clinical diagnostics, and pharmacokinetic studies.