Analysis of Cannabinoids and their Metabolites in Human Urine Using the Agilent Chem Elut S Plate by LC/MS/MS

Importance of the Topic

The rising medicinal and recreational use of cannabis has increased the need for reliable monitoring of cannabinoids and their metabolites in biological fluids.
Accurate urine analysis is critical for forensic toxicology, clinical compliance, and workplace drug testing.

Objectives and Study Overview

This study aimed to develop and validate a high-throughput LC/MS/MS method to quantify THC, CBD, CBN, 11-hydroxy-Δ9-THC and 11-nor-9-carboxy-Δ9-THC in human urine using the Agilent Chem Elut S 96-well plate.
The goal was to achieve sensitivity down to 2 ng/mL, a broad linear range, and robust precision and accuracy suitable for forensic applications.

Methodology and Instrumentation

Urine samples (150 µL) were fortified with internal standards and 5% isopropanol to prevent nonspecific binding, then hydrolyzed with IMCSzyme E1F β-glucuronidase.
Supported liquid extraction was performed on an Agilent Chem Elut S 400 µL 96-well plate using two rinses of 9:1 hexane/ethyl acetate to maximize recovery.
Extracts were dried under nitrogen at 30 °C and reconstituted in acetonitrile/methanol prior to analysis.
Chromatography used an Agilent ZORBAX Eclipse Plus C18 column with gradient elution, and detection employed an Agilent 6490 triple quadrupole mass spectrometer in dynamic MRM mode with positive electrospray ionization.

Key Results and Discussion

The method showed excellent linearity (R2 > 0.99) over 2–1000 ng/mL for all analytes.
LLOQ at 2 ng/mL yielded signal-to-noise ratios above 10 and matrix blank interference below 20% of the analyte response.
Accuracy at QC levels (2, 50, 1000 ng/mL) ranged from 93% to 111%, and precision RSDs were ≤15% (≤20% at LLOQ).
Combined hydrolysis and extraction recovery for THC-COOH glucuronide was 81% (RSD 1.6%).
Adding isopropanol and using glassware effectively minimized nonspecific adsorption, particularly for hydrophobic cannabinoids.

Benefits and Practical Applications

This protocol offers rapid, automated sample preparation with reduced manual steps and improved reproducibility compared to traditional liquid–liquid extraction.
High-throughput compatibility makes it suitable for forensic and clinical laboratories requiring reliable quantitation of cannabinoids in urine.

Future Trends and Potential Applications

Integration with robotic liquid handling and on-line extraction could further increase throughput and consistency.
Extending the approach to other biological matrices such as plasma or oral fluid would broaden its clinical and forensic utility.
Expansion of the analyte panel to include minor cannabinoids and synthetic analogues can support evolving drug monitoring needs.

Conclusion

The validated LC/MS/MS workflow combining IMCSzyme digestion and Chem Elut S extraction delivers a sensitive, precise, and efficient solution for quantifying major cannabinoids and their metabolites in human urine.

Used Instrumentation

• Agilent 1290 Infinity II LC system
• Agilent 6490 triple quadrupole mass spectrometer
• Agilent Chem Elut S 400 µL 96-well plate
• IMCSzyme E1F β-glucuronidase kit

Reference

• Binnian W. et al. Analysis of Cannabinoids and Their Metabolites in Human Urine, Analytical Chemistry, 2015, 87(20), 10183–10187.
• IMCSzyme E1F kit product information, IMCS Tips website.