Automated determination of THC contents in human urine

Importance of the Topic

Monitoring Δ9-tetrahydrocannabinol (THC) and its key metabolites in human urine is critical for forensic toxicology, workplace drug testing, therapeutic drug monitoring and pharmacokinetic studies. Reliable quantification at low nanogram levels ensures accurate interpretation in clinical and legal contexts.

Objectives and Study Overview

This study presents an automated workflow for the extraction and quantification of THC, 11-hydroxy-THC (THC-OH) and THC-COOH from spiked human urine samples. By integrating an automated solid-phase extraction (SPE) system with subsequent high-performance liquid chromatography–mass spectrometry (HPLC-MS), the aim was to streamline sample preparation, increase throughput and improve recovery rates relative to manual SPE.

Methodology

Sample Preparation and Hydrolysis:

• Human urine was spiked to achieve 10 ng/mL of each analyte.
• Glucuronide conjugates were cleaved by adding NaOH (10 M), incubation at 60 °C for 15 min, neutralization with acetic acid and pH adjustment to 6–7 with ammonium acetate buffer.

Automated SPE on FREESTYLE™ System:

• Cartridge: CHROMABOND® HR-X, 200 mg, 3 mL capacity.
• Conditioning: Methanol, water, ammonium acetate buffer at 2 mL/min.
• Loading: Sample applied at 1–2 mL/min.
• Wash: Methanol–water (30:70 v/v), drying for 10 min.
• Elution: Hexane–ethyl acetate–acetic acid (75:25:1 v/v/v).

Eluent Exchange:
Eluates evaporated under N2 at 40 °C, reconstituted in methanol (1 mL).

Used Instrumentation

• Automated SPE: FREESTYLE™ SPE system (LCTech) with 6-solvent capability and rack modules for SPE cartridges and sample vials.
• Chromatography: Dionex Ultimate 3200 HPLC (HPG) with Macherey-Nagel NUCLEOSHELL® RP 18 column (50×2 mm, 2.7 µm).
• Detection: ABSciex API 3200 MS in positive ESI mode, MRM transitions for quantifier and qualifier ions.

Main Results and Discussion

Chromatographic runtime was 7.5 min, achieving clear separation of THC, THC-OH and THC-COOH. Two MS/MS transitions per analyte ensured confident identification. Recovery rates (mean ± SD, n=3) were:

• THC: manual SPE 80 ± 3 %; automated SPE 90 ± 9 %.
• THC-OH: manual 77 ± 10 %; automated 80 ± 6 %.
• THC-COOH: manual 98 ± 8 %; automated 93 ± 8 %.

Automated SPE matched or exceeded manual recovery, demonstrating robustness and reproducibility.

Benefits and Practical Applications

The fully automated SPE protocol reduces hands-on time, minimizes variability and allows unsupervised high-throughput processing. Combined with fast HPLC-MS, this approach is ideal for clinical laboratories, forensic facilities and research centers requiring large-scale cannabinoid testing.

Future Trends and Applications

Advances may include integration with online LC-MS systems for direct sample analysis, expansion to other drug classes, and implementation of real-time data processing through artificial intelligence. Miniaturized cartridges and green solvents could further enhance efficiency and sustainability.

Conclusion

The automated FREESTYLE™ SPE coupled with HPLC-MS provides a reliable, fast and high-throughput method for THC and metabolite quantification in urine, outperforming manual protocols in recovery and consistency.

Reference

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