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

Significance of the Topic

Efficient and reliable monitoring of tetrahydrocannabinol (THC) and its metabolites in biological matrices is critical for forensic toxicology, clinical investigations, and workplace drug testing. A unified sample preparation approach can streamline workflows, minimize analyte losses, and improve laboratory throughput.

Objectives and Study Overview

The primary aim was to develop a single solid-phase extraction (SPE) protocol using Oasis MAX cartridges suitable for both urine and blood samples. This method needed to be compatible with either UPLC/MS/MS or GC/MS/MS analysis and deliver high sensitivity, reproducibility, and linearity for THC, hydroxy-THC (OH-THC), and carboxy-THC (COOH-THC).

Methodology

The procedure employs a mixed-mode strong anion-exchange Oasis MAX sorbent offering reversed-phase retention of neutral analytes and ionic retention of acidic metabolites. Blood samples were precipitated with acetonitrile, diluted in aqueous ammonia, and loaded directly onto SPE without evaporation. Urine samples underwent alkaline hydrolysis, pH adjustment, buffer addition, and organic dilution before SPE. Elution was performed with a hexane-ethyl acetate-acetic acid mixture, followed by solvent evaporation and reconstitution tailored to the chosen analysis technique. For UPLC/MS/MS, an ACQUITY BEH C18 column with a 60–90% acetonitrile gradient and electrospray ionization (ESI+) for THC/OH-THC and ESI- for COOH-THC was used. GC/MS/MS analysis used RTX-5MS chromatography and silylation with BSTFA/TMCS.

Instrumentation Used

• Waters ACQUITY UPLC system with BEH C18 column
• Waters Quattro Premier XE tandem mass spectrometer
• Waters Quattro micro GC/MS/MS system
• Oasis MAX SPE cartridges
• Silylation oven and autosampler for GC derivatization

Main Results and Discussion

Both LC/MS/MS and GC/MS/MS methods achieved linear calibration curves from 0 to 100 ng/mL with coefficients of determination (r2) exceeding 0.99. Urine extractions yielded recoveries of 92% (7% RSD) and blood extractions showed overall recoveries of 78% (7% RSD), with true SPE recovery near 90%. Matrix effects remained below 10% ion suppression. The limit of quantitation was conservatively estimated at 0.5 ng/mL. The method demonstrated robustness, processing hundreds of samples without instrument maintenance.

Benefits and Practical Applications

• Single SPE workflow for two biological matrices reduces complexity and development time
• Avoidance of evaporation steps minimizes analyte loss and simplifies blood processing
• Compatibility with both LC/MS/MS and GC/MS/MS meets diverse laboratory needs
• High sensitivity and reproducibility support forensic, clinical, and QA/QC testing

Future Trends and Potential Uses

The protocol can be adapted for automated high-throughput SPE platforms. Coupling with high-resolution mass spectrometry may enable broader profiling of cannabinoids, including emerging synthetic analogs and novel metabolites. Integration with rapid screening tools could further enhance forensic and clinical applications.

Conclusion

The developed Oasis MAX SPE protocol provides a streamlined, versatile, and high-performance solution for quantifying THC and its primary metabolites in urine and blood via UPLC/MS/MS or GC/MS/MS. It offers significant advantages in simplicity, sensitivity, and robustness for analytical laboratories.