UPLC Separation for the Analysis of Cannabinoid Content in Cannabis Flower and Extracts

Significance of the Topic

The increasing legalization of cannabis has driven the need for fast, reliable methods to quantify both major and minor cannabinoids in flower and extract products. Accurate potency analysis supports producers, regulators, and consumers by ensuring product consistency, safety, and compliance.

Objectives and Study Overview

This work adapts a previously published isocratic HPLC method for the separation of 16 cannabinoids into a UPLC format. The goal is to enhance sample throughput and reduce solvent usage while retaining the selectivity and linearity of the original method.

Methodology and Instrumentation

• Instrumentation used:
  • ACQUITY UPLC H-Class System
  • ACQUITY UPLC PDA Detector
  • Empower 3 Data Software
  • CORTECS UPLC Shield RP18 column (2.1 × 100 mm, 1.6 µm)
• Chromatographic conditions:
  • Mobile phase A: water + 0.1% TFA; mobile phase B: acetonitrile; isocratic 41:59
  • Flow rate: 0.7 mL/min; column temperature: 35 °C
  • Detection wavelength: 228 nm (4.8 nm resolution); injection volume: 0.7 µL
• Samples and standards:
  • DEA-exempt cannabinoid standards prepared in methanol, serially diluted (0.004–1 mg/mL)
  • Flower samples extracted in acetonitrile or ethanol; concentrates extracted in isopropanol; all filtered through 0.22 µm tips

Main Results and Discussion

The UPLC method achieved baseline separation of 16 cannabinoids in 10.5 minutes with all resolution values (Rs) exceeding 2.0. Calibration for CBD and Δ9-THC demonstrated excellent linearity (R² ≥ 0.9998) across ten concentration levels. Compared with the original HPLC procedure, UPLC delivered a 2.5-fold increase in daily sample throughput and an 86% reduction in solvent consumption.

Benefits and Practical Applications

This rapid, high-resolution UPLC protocol offers significant solvent savings and increased productivity, making it ideal for routine potency testing and quality control in cannabis laboratories.

Future Trends and Applications

Further advancements may include coupling UPLC separations with high-resolution mass spectrometry, implementing automated sample preparation, and expanding analysis to emerging minor cannabinoids for comprehensive profiling.

Conclusion

The transition to UPLC provides a robust, efficient, and sustainable approach for quantifying complex cannabinoid mixtures, meeting modern analytical requirements for speed, sensitivity, and data quality.

References

• AJ Aubin, CE Layton. Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed Phase Isocratic HPLC Method. Waters Application Note 720006426EN (2018).
• MM Radwan et al. Natural Cannabinoids of Cannabis and Methods of Analysis. In: Cannabis sativa L. – Botany and Biotechnology. Springer, Cham (2017).
• CE Layton, AJ Aubin. Method Validation for Assay Determination of Cannabidiol Isolates. Journal of Liquid Chromatography & Related Technologies, 41(3) (2018).
• CE Layton, AJ Aubin. Setting the Standard: Considerations When Handling Cannabinoid Reference Standard Preparations Used for Potency Determination. Cannabis Science and Technology, 1(3) (2018).
• USP General Chapter <621> System Suitability. USP40-NF35 Supplement.
• CE Layton. Beginner’s Guide to Preparative Liquid Chromatography. Waters (715005428) (2017).