Extraction of THC-COOH from Urine Using ISOLUTE® HAX Columns

Importance of the Topic

Reliable detection of the THC-COOH metabolite in urine is essential for clinical toxicology, workplace drug testing, and forensic investigations. This analytical workflow ensures sensitive, selective extraction and quantitation of a key marijuana biomarker in complex biological matrices.

Objectives and Study Overview

The primary goal was to develop and validate a solid-phase extraction method for isolating THC-COOH from urine, followed by gas chromatography–mass spectrometry (GC-MS) analysis. Specific aims included:

• Maximizing analyte recovery (>75%)
• Removing endogenous interferences
• Ensuring compatibility with GC-MS derivatization requirements

Methodology and Instrumentation

The extraction procedure employs mixed-mode nonpolar/anionic exchange cartridges (ISOLUTE HAX, 200 mg/10 mL). Key steps:

1. Pre-treatment: 3 mL urine + 100 µL internal standard + hydrolysis with 0.1 mL 10 M NaOH, 60 °C for 15 min, neutralization with acetic acid.
2. Conditioning: 1 mL methanol at 2 mL/min.
3. Equilibration: 1 mL deionized water at 2 mL/min.
4. Sample loading: 3 mL processed urine at 2 mL/min.
5. Interference elution: sequential rinses with ammonium acetate buffers (0.3 M, pH 8; then 20 mM) and methanol.
6. Analyte elution: 1 mL methanol/ethyl acetate/acetic acid (48:50:2) at 1 mL/min.

Used Instrumentation

The purified extract is derivatized for GC-MS analysis. Instrument details:

• GC column: DB-5 capillary, 15 m × 0.25 mm I.D., 0.25 µm film.
• Oven program: 180 °C (1 min), ramp 20 °C/min to 280 °C (1 min).
• Detection: Mass spectrometer in selected ion monitoring (SIM) mode.

Key Results and Discussion

The mixed-mode extraction achieved consistent THC-COOH recoveries above 75% across multiple urine samples, with effective removal of salts and endogenous matrix components. The use of anion exchange combined with nonpolar retention provided high selectivity, reducing background noise in the final GC-MS chromatograms.

Instrument performance demonstrated sharp, well-resolved peaks and reproducible signal intensities. The method tolerated variable urine ionic strength and delivered stable quantitation over repeated cycles, confirming robustness.

Benefits and Practical Applications

This workflow offers:

• High selectivity and sensitivity for THC-COOH in forensic and clinical settings.
• Compatibility with routine GC-MS instrumentation.
• Efficient sample cleanup for high-throughput laboratories.
• Cost-effective cartridge format suitable for large batch analyses.

Future Trends and Opportunities

Advancements may include:

• Automation of cartridge processing for even higher throughput.
• Integration with tandem mass spectrometry (GC-MS/MS) for enhanced selectivity.
• Extension to other cannabinoid metabolites and novel psychoactive substances.
• Development of miniaturized or on-site extraction devices.

Conclusion

The described ISOLUTE HAX extraction paired with GC-MS provides a reliable, high-recovery method for quantifying THC-COOH in urine. Its robustness and selectivity make it well suited to compliance testing, forensic analysis, and clinical toxicology laboratories.

References

• Biotage. Application Note IST1035A.V.1, 2020.