Purification of Cannabinoids from a Cannabis sativa Extract

Importance of the Topic

The purification of individual cannabinoids from complex Cannabis sativa extracts has become critical as consumer products in food, cosmetics and healthcare expand. Reliable isolation of single compounds is needed to generate high-quality reference standards, ensure consistency in pharmaceutical development and support regulatory compliance in quality control laboratories.

Objectives and Study Overview

This study aims to demonstrate an integrated analytical and preparative workflow for the selective isolation of multiple cannabinoids from a cannabidiol-rich hemp oil extract. The goals are:

• To develop and optimize an LC–MS method for analytical scouting of cannabinoid profiles.
• To scale the method to preparative conditions for fraction collection.
• To apply mass-based triggers for high-specificity fractionation.
• To assess purity and recovery of isolated fractions.

Methodology

A reversed-phase LC gradient was established on an analytical column (4.6 × 150 mm, C18, 5 µm) using 0.1 % formic acid in water (A) and 0.05 % formic acid in methanol (B). The gradient ramped from 60 % B to 95 % B over 9 minutes at 1.5 mL/min. Detection combined UV at 230 nm with single-ion-monitoring (SIM) in both positive and negative modes to target seven characteristic m/z values for 13 known cannabinoids. After analytical scouting, the same gradient was transferred unchanged to a preparative column (21.2 × 150 mm, C18, 5 µm) at 31.9 mL/min. Mass-based fraction collection was activated via SIM signals of deprotonated and sodium-adduct ions.

Used Instrumentation

Key hardware components:

• Agilent 1290 Infinity II Autoscale Preparative LC/MSD System with two flow paths (analytical and preparative) sharing a single MSD and combined sampler/fraction collector.
• Preparative binary pump, open-bed sampler/collector, and variable-wavelength detector (230 nm).
• Diode array detector, preparative column compartment, preparative valve drive, MS flow modulator.
• Agilent InfinityLab LC/MSD XT with Jet Stream electrospray ion source.

Key Results and Discussion

Analytical scouting revealed that UV detection alone missed several minor cannabinoids, whereas SIM-MS uncovered additional components. The main UV peak at 8.5 min (CBD) and a secondary peak at 7.2 min (THV) were confirmed by m/z 313.2 (negative SIM) and 285.2 (negative SIM) respectively. Preparative runs showed a slight retention shift due to reduced dwell volume but preserved resolution. Ten fractions were collected by mass-trigger at the predefined m/z values; only two were strongly UV-active, demonstrating the advantage of MS-based collection for low-abundance targets. Reanalysis of the CBD fraction yielded 92–97 % recovery and 95 % UV-based purity. MSD reanalysis identified trace co-eluting impurities not apparent in the UV trace, highlighting when additional cleanup may be required.

Benefits and Practical Applications

This integrated system offers several advantages for cannabinoid purification workflows:

• Bench-space efficiency and cost savings by sharing one MSD across two flow paths.
• Seamless method transfer from analytical scouting to preparative fractionation.
• Mass-based triggers ensure selective collection of targeted compounds, including those undetectable by UV.
• Automated fraction reanalysis simplifies quality assessment without manual vial transfers.

Future Trends and Potential Applications

Continued growth in cannabinoid research and commercialization will drive demand for robust, automated purification methods. Future developments may include:

• Enhanced stationary phases for faster separations and improved resolution of isomers.
• Integration of orthogonal detection modes (e.g., UV–fluorescence or high-resolution MS) for comprehensive profiling.
• Miniaturized preparative systems for on-demand reference standard production in clinical laboratories.
• Expanded software capabilities for predictive scale-up and real-time fraction monitoring.

Conclusion

The Agilent 1290 Infinity II Autoscale Preparative LC/MSD workflow successfully combined analytical scouting, mass-based fraction collection and fraction reanalysis in a single platform. This approach achieves high recovery and purity of cannabinoids from hemp oil extracts, improves selectivity over UV-only methods, and streamlines the production of reference compounds for quality control and research applications.

References

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2. Cannabis Act. Statutes of Canada 2018, c. 16. Current to 20 June 2019.
3. State Medical Marijuana Laws. Natl Conf State Legislatures. 2 July 2019.
4. FDA News Release. U.S. Food and Drug Administration; 26 June 2018.
5. Hazekamp A, et al. Chromatographic and Spectroscopic Data of Cannabinoids from Cannabis sativa L. J Liq Chromatogr Relat Technol. 2005;28(15):2361–2382.