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

Importance of the Topic

The selective extraction of THC-COOH from urine is critical in forensic and clinical analysis to confirm cannabis exposure. Reliable sample preparation ensures accurate quantification by minimizing matrix effects and enhancing sensitivity.

Objectives and Study Overview

This application note describes the development of a mixed-mode solid-phase extraction method using ISOLUTE HAX columns to isolate the primary THC metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), from urine samples. The protocol aims for robust analyte retention, efficient removal of co-extracted impurities, and recoveries above 75%.

Methodology and Protocol

Urine samples (3 mL) are spiked with an internal standard (100 µL) and subjected to alkaline hydrolysis (10 M NaOH, 60 °C, 15 min). After cooling and acidification with glacial acetic acid, SPE cartridges are conditioned with methanol and equilibrated with deionized water. Samples are loaded at 2 mL/min; interferences are eluted sequentially using a 0.3 M ammonium acetate buffer (pH 8), a 20 mM ammonium acetate buffer, and methanol. THC-COOH is finally eluted with a mixture of methanol/ethyl acetate/acetic acid (48:50:2).

Used Instrumentation

Gas chromatograph equipped with a DB-5 capillary column (15 m × 0.25 mm I.D., 0.25 µm) coupled to a mass spectrometer. The temperature program starts at 180 °C (1 min), ramps at 20 °C/min to 280 °C (1 min), with MS detection for sensitive analyte confirmation.

Main Results and Discussion

The SPE procedure consistently delivers THC-COOH recoveries exceeding 75% and substantially reduces matrix interferences. The mixed non-polar and anion-exchange mechanism provides high selectivity, leading to clean chromatograms and reproducible quantitation.

Practical Benefits and Applications

• Forensic toxicology: confirms marijuana use in legal investigations and workplace drug testing.
• Clinical toxicology: monitors patient compliance and potential intoxication in therapeutic contexts.
• Anti-doping control: screens athletes for cannabinoid metabolites with reliable sensitivity.

Future Trends and Possibilities

Automation of SPE workflows and integration with high-resolution mass spectrometry may further enhance throughput and analytical performance. Development of novel sorbent chemistries could improve selectivity and recovery for a broader range of cannabinoid metabolites.

Conclusion

The ISOLUTE HAX SPE protocol offers a robust, high-recovery approach for extracting THC-COOH from urine, supporting accurate GC-MS analysis in forensic, clinical, and doping-control laboratories.

References

Biotage. Application Note IST1035A.V.1. ©2020 Biotage.