(C)an(n)alyze: determination of 16 cannabinoids inside flowers, oils and seeds

Importance of the topic

The increasing use of Cannabis sativa L. in pharmaceutical, nutraceutical and industrial contexts drives a need for reliable methods to characterize its bioactive cannabinoids. Accurate profiling of these compounds supports quality control, regulatory compliance and therapeutic research, ensuring consistent potency and safety of cannabis-derived products.

Study objectives and overview

This work aimed to extend and accelerate the German Pharmacopeia HPLC method by including 16 cannabinoids for simultaneous qualification and quantification in three sample types: flowers, oils and seeds. Key goals were to shorten run time, simplify eluent acidity and validate the method against ICH Q2(R1) and AOAC performance criteria.

Used instrumentation

• HPLC system: KNAUER AZURA® with P 6.1 L LPG pump, AS 6.1 L autosampler, CT 2.1 column thermostat, MWD 2.1 L diode‐array detector
• Column: Eurospher II C18P, 100×4.6 mm, 3 µm
• Detection wavelengths: 228 nm and 306 nm

Methodology

The optimized gradient employed water (pH 2.2 with phosphoric acid) and acetonitrile, with a total run time of 22 min. Sample preparation followed an ultrasonic extraction in 80 % methanol, filtration through 0.20 µm PTFE and direct injection of 10 µL. Validation parameters included selectivity, linearity (0.15–75 µg/mL), repeatability and recovery.

Main results and discussion

The method achieved baseline separation of 16 cannabinoids in under 20 min. Calibration curves exhibited linearity coefficients > 0.999. Repeatability across three concentration levels yielded RSD values below 1 % for all detected analytes, meeting AOAC criteria. Recovery studies at 0.20 and 0.40 µg/mL produced rates between 95 % and 105 %. In flower and oil matrices, 11 and 8 cannabinoids respectively were quantified; seeds showed no detectable cannabinoids above LOQ. Δ9-THC quantification required absence of its degradation product CBNA, distinguishable at 306 nm.

Benefits and practical applications

• Rapid screening of multiple cannabinoids in diverse hemp matrices
• Compliance with pharmacopoeial and regulatory standards for medicinal cannabis
• High precision supports routine quality control in pharmaceutical and nutraceutical labs
• Adaptable to industrial QA/QC for food and cosmetic products containing hemp extracts

Future trends and applications

Advancements may include coupling with mass spectrometry for enhanced sensitivity and structural confirmation, miniaturization for high‐throughput screening, and integration into automated platforms for real‐time monitoring of cultivation and extraction processes. Expanding the panel to novel minor cannabinoids and metabolites will further support research into therapeutic effects.

Conclusion

A fast, robust and fully validated HPLC‐DAD method was developed for the simultaneous analysis of 16 cannabinoids, meeting international guidelines. Its high throughput and accuracy make it a valuable tool for ensuring the quality and safety of cannabis‐based products.

References

• Deutsches Arzneibuch 2017 (DAB), General Chapter, 2018.
• ICH Q2(R1) Validation of Analytical Procedures, 1995.
• AOAC Standard Method Performance Requirements for Cannabis, 2016.
• Mudge E.M., Murch S.J., Brown P.N., Anal. Bioanal. Chem. 409 (2017) 3153–3163.