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

Importance of the Topic

Determination of Δ9-tetrahydrocannabinol (THC) and its primary metabolites hydroxy-THC (OH-THC) and carboxy-THC (COOH-THC) in biological fluids is essential for forensic, clinical and workplace drug testing. Blood analysis indicates recent cannabis use and potential impairment, while urine screening reveals past exposure through detection of longer-lived COOH-THC. Developing a reliable, fast and universal sample preparation method for both matrices enhances laboratory efficiency and data quality.

Objectives and Study Overview

This work aimed to establish a single solid-phase extraction (SPE) protocol for both urine and blood samples that is compatible with either ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) or gas chromatography/tandem mass spectrometry (GC/MS/MS). Key goals included:

• Unified SPE workflow for all analytes and matrices
• Minimal sample pre-preparation time
• High recoveries, low matrix effects and linear response over 0.5–100 ng/mL
• Demonstration of rapid UPLC separation and complementary GC/MS/MS performance

Used Instrumentation

• ACQUITY UPLC system with BEH C18 column (2.1 × 100 mm, 1.7 µm)
• Quattro Premier XE tandem quadrupole mass spectrometer (ESI+ and ESI– modes)
• Quattro micro GC/MS/MS system with RTX-5MS column (30 m × 0.025 mm × 0.25 µm)
• Oasis MAX mixed-mode strong anion-exchange SPE cartridges (60 mg, 3 cc)

Methodology

Sample pre-treatment:

• Blood: proteins precipitated with acetonitrile, centrifugation, dilution to 40% organic before SPE
• Urine: alkaline hydrolysis of glucuronides, pH adjustment and dilution in phosphate buffer with acetonitrile

SPE protocol (Oasis MAX):

1. Condition and equilibrate cartridge
2. Load prepared sample
3. Wash with water/methanol and hexane
4. Air dry under vacuum
5. Elute with hexane:ethyl acetate:acetic acid (49:49:2)
6. Evaporate and reconstitute in appropriate solvent (ethyl acetate for GC, acetonitrile/water for LC)

Chromatographic conditions:

• UPLC: gradient from 60% to 90% acetonitrile with 0.1% formic acid over 4 min, flow 400 µL/min
• GC: temperature program from 100 °C to 300 °C, helium carrier, splitless injection

MS/MS detection:

• Multiple reaction monitoring (MRM) for THC, OH-THC and COOH-THC with deuterated internal standards

Key Results and Discussion

Both UPLC/MS/MS and GC/MS/MS methods delivered consistent linearity (r² > 0.99) for all analytes in blood and urine over 0.5–100 ng/mL. Method precision (RSD) ranged from 6% to 14% at low-level spikes (0.5 ng/mL). Recoveries averaged 90% in urine and 78%–92% in blood (the latter influenced by protein precipitation), with ion suppression below 10%. Limits of quantitation of 0.5 ng/mL were conservatively established for all analytes. The mixed-mode Oasis MAX sorbent maintained high retention even with 40% organic content, eliminating evaporation steps and reducing sample handling losses.

Benefits and Practical Applications

• Single SPE protocol for both matrices and both analytical platforms
• Reduced sample preparation time and minimized analyte losses
• High throughput capability supported by rapid UPLC separation and reliable GC/MS/MS analysis
• Enhanced reproducibility and sensitivity suitable for forensic and clinical laboratories

Future Trends and Potential Applications

Ongoing developments in SPE sorbent chemistries and fully automated sample preparation systems will further streamline workflows. Coupling high-resolution accurate-mass MS to rapid UPLC promises improved selectivity and the ability to screen additional minor cannabinoids. Extending the protocol to alternative matrices (e.g., oral fluid, hair) and integrating on-line SPE could support near real-time monitoring in clinical and roadside testing.

Conclusion

A robust, universal SPE approach using Oasis MAX cartridges has been demonstrated for reliable quantitation of THC and its metabolites in urine and blood via both UPLC/MS/MS and GC/MS/MS. The unified workflow reduces preparation steps, delivers high recoveries and consistent low-level performance, making it well suited for forensic, clinical and industrial laboratories seeking efficient cannabinoid analysis.

References

• Waters Corporation. Optimized SPE for UPLC/MS/MS and GC/MS/MS Determination of THC and Its Metabolites in Urine and Blood. Application Note 720003738EN, 2010.