Improved Extraction of THC and its Metabolites from Oral Fluid Using Oasis PRiME HLB Solid Phase Extraction (SPE) and a UPLC CORTECS C18 Column

Importance of the Topic

Cannabis use continues to expand for both medical and recreational purposes, driving the need for reliable analytical methods to detect Δ-9-tetrahydrocannabinol (THC) and its major metabolites in biological fluids. Oral fluid has become a favored matrix for roadside testing and impairment assessment due to its non-invasive collection, ease of supervision, and closer correlation with recent use and pharmacological effects compared to urine.

Objectives and Study Overview

The study aimed to develop a semi-validated workflow for the extraction and quantification of THC, 11-hydroxy-THC (THC-OH), and 11-nor-9-carboxy-THC (THC-COOH) in oral fluid. Key goals included:

• Simplifying sample preparation by eliminating conditioning and evaporation steps.
• Providing linear, accurate, and precise quantification down to low nanogram-per-milliliter levels.
• Minimizing matrix effects that can compromise mass spectrometric detection.
• Ensuring throughput compatible with forensic and clinical laboratories.

Methodology and Instrumentation

Sample Collection and Pre-Treatment:
The Quantisal oral fluid device was used to collect approximately 1 mL of fluid, stabilized with proprietary buffer. Samples were spiked with deuterated internal standards and acidified with phosphoric acid prior to solid phase extraction (SPE).

SPE Protocol:
Oasis PRiME HLB µElution Plates enabled a three-step workflow without conditioning or equilibration:

• Load 710 µL of treated sample directly onto the SPE plate.
• Wash twice with 250 µL of 5% ammonia in 25:75 methanol:water to remove matrix interferences.
• Elute with two 25 µL aliquots of 90:10 acetonitrile:methanol, then dilute with water for direct injection.

Chromatographic and Detection Conditions:
Analysis was performed on an ACQUITY I-Class UPLC equipped with a CORTECS C18 column (1.6 µm, 2.1 × 100 mm) held at 40 °C. A water/formic acid–acetonitrile/formic acid gradient delivered baseline separation of all analytes within three minutes. Detection employed Xevo TQ-S triple quadrupole MS in positive electrospray mode, monitoring two multiple reaction monitoring (MRM) transitions per compound.

Main Results and Discussion

Chromatography and Sensitivity:
All three compounds exhibited sharp peaks (<1.8 s width) and eluted by 3 minutes. The method achieved lower limits of quantification of 0.05 ng/mL for THC and 0.1 ng/mL for its metabolites.

Recovery and Matrix Effects:
Extraction recoveries exceeded 75% for each analyte with relative standard deviations below 6%. Matrix effects were below 10% after optimizing the wash step with ammoniated solvent, demonstrating robust cleanup of oral fluid constituents.

Quantitative Performance:
Calibration curves were linear (r2>0.999) over four orders of magnitude (0.05–100 ng/mL). Quality control samples at multiple concentration levels showed accuracies within 10% of nominal values and precisions under 3% RSD, meeting regulatory guidelines for bioanalytical methods.

Benefits and Practical Applications

• Non-invasive, easily supervised sampling suitable for roadside and workplace testing.
• High-throughput sample preparation without evaporation or reconstitution.
• Rapid UPLC-MS/MS analysis enables same-day turnaround.
• Reliable quantification at or below 2 ng/mL cutoff concentrations for forensic thresholds.

Future Trends and Potential Applications

• Integration with automated sample preparation platforms to further boost throughput.
• Expansion to other emerging cannabinoids and synthetic analogs in saliva.
• Combination with on-site screening tools for preliminary results followed by confirmatory UPLC-MS/MS.
• Application in epidemiological studies to monitor real-time cannabis consumption patterns.

Conclusion

The described method leverages Oasis PRiME HLB µElution SPE and rapid UPLC-MS/MS analysis to achieve consistent, sensitive, and precise quantification of THC and its key metabolites in oral fluid. The streamlined workflow and excellent analytical performance make it suitable for forensic toxicology, workplace testing, and clinical research, addressing the growing demand for reliable non-invasive drug testing.

References

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