Separation of 16 Cannabinoids Using the Agilent 1260 Infinity II LC System

Significance of the Topic

The increasing demand for cannabis-derived products requires robust analytical methods for profiling cannabinoids.
Quantifying a broad range of cannabinoids ensures product consistency, regulatory compliance, and quality control across cultivation, processing, and formulation.

Objectives and Study Overview

The study aimed to develop and validate an efficient liquid chromatography method capable of separating and quantifying 16 major cannabinoids in under 12 minutes.
Key goals included achieving baseline resolution for all analytes, establishing calibration linearity, and assessing method accuracy and precision.

Methodology and Instrumentation

• Chromatographic Conditions
  • Column: Poroshell 120 EC-C18, 3.0×150 mm, 2.7 µm with matching guard column
  • Mobile phases: 5 mM ammonium formate + 0.1% formic acid (aqueous) and 0.1% formic acid in acetonitrile
  • Gradient: 75% to 90% organic over 11 min at 0.8 mL/min
  • Column temperature: 30 °C; injection volume: 5 µL; detection at 230 nm
• Instrumentation Used
  • Agilent 1260 Infinity II binary pump, vialsampler, multicolumn thermostat, diode array detector
  • Agilent OpenLab CDS Workstation software Version 2.7
• Sample Preparation
  • Plant material: homogenize 200 mg in methanol, centrifuge, dilute, filter
  • Oils/concentrates: dilute 100 µL in ethanol, filter, further dilute in methanol
• Calibration and Validation
  • Five-point external calibration from 0.5 to 50 µg/mL
  • Linearity over three days with R² ≥ 0.999
  • LOD and LOQ calculated as 3.3σ and 10σ of low-level standards

Main Results and Discussion

The optimized method achieved baseline separation (resolution > 1.5) of all 16 cannabinoids within 12 min, including challenging pairs such as Δ8-THC/Δ9-THC and CBD/CBDA.
Calibration curves demonstrated excellent linearity (R² ≥ 0.9998) across analytes and days.
Accuracy ranged from 95% to 120% depending on concentration, while precision (RSD) remained below 5% for most analytes at higher levels.
LOD values spanned 0.02 to 0.14 µg/mL, indicating high sensitivity suitable for regulatory testing.

Benefits and Practical Applications

• High-throughput screening in testing laboratories
• Applicability to diverse matrices: plant material, oils, tinctures, resins
• Compliance with regulatory requirements for comprehensive cannabinoid profiling

Future Trends and Opportunities

• Extension to minor cannabinoids by exploiting chromatographic capacity
• Coupling with mass spectrometry for enhanced specificity
• Automation of sample preparation and data reporting for higher throughput

Conclusion

The Agilent 1260 Infinity II LC method delivers rapid, reliable separation and quantitation of 16 cannabinoids, supporting quality control and regulatory compliance across the cannabis industry.

References

• ElSohly, M.; Gul, W. Constituents of Cannabis Sativa. In Handbook of Cannabis; Oxford University Press, 2014.
• Hillig, K. W.; Mahlberg, P. G. A Chemotaxonomic Analysis of Cannabinoid Variation in Cannabis (Cannabaceae). Am. J. Bot. 2004, 91, 966–975.