UHPLC Enables Rapid Quantitation of Native Cannabinoids

A highly sensitive method for the quantitation of 11 major cannabinoids from cannabis samples has been outlined by researchers at the University of Mississippi. The team sought to identify an efficient strategy for extracting cannabinoids and trace components from a variety of cannabis samples. They were also keen to highlight the benefits of ultra-high-performance liquid chromatography-based (UHPLC) analysis for the accurate quantitation of cannabinoids.  Their findings were published in Planta Medica.  

The challenges of common cannabinoid analysis methods 

When asked about the motivation behind this research, corresponding author Dr Mahmoud ElSohly, Research Professor in the research Institute of Pharmaceutical Sciences and Professor of Pharmaceutics, gave a straightforward response “There is always a need to improve analytical methods for the quantitation of different cannabinoids in cannabis products.” Having headed up the only U.S. government licensed cannabis cultivation and research program at the University of Mississippi since the early 1980’s, Dr ElSohly understands this better than most.  

Gas chromatography (GC) and high-pressure liquid chromatography (HPLC) are probably the two most common methods used for cannabinoid quantitation. However, both come with inherent challenges. GC can be run quickly and efficiently but it is difficult to directly determine acidic cannabinoids such as tetrahydrocannabinolic acid (THCA) and cannabidiolic Acid (CBDA) as they are decarboxylated by the heated injection port on the instrument. This can be remedied by adding a deritization step, but this requires additional time and resources per run. On the other hand, HPLC is suitable for analyzing cannabinoids in all native forms, including acidic, but is slow to run. The authors explain “… the total run time of one analysis on HPLC including the time of separation and column re-equilibration is usually more than 25 minutes”. UHPLC suffers neither of these drawbacks with run times of 10 minutes in this study and, no requirement for heating. 

Alongside identifying an effective, accurate method for cannabinoid quantitation, the researchers sought to determine the optimal extraction solvents for preparing cannabis samples for analysis. The authors highlight how many different solvents have been used in previous studies including ethanol, methanol, chloroform, hexane, petroleum ether and various mixtures of these chemicals. In many of these earlier studies, recovery rates were not reported. They go on to explain that where all details have been recorded, a mixture of methanol and chloroform delivered recovery rates of above 90% for major components, but below 85% for the cannabinoids cannabigerolic acid (CBGA), cannabigerol (CBG) and delta-8-tetrahyrocannabinol (Δ8-THC). As these published recovery rates are relatively low, the team believed there was scope for the optimization of extraction solvents to enhance results.  

Optimizing cannabinoid extraction and detection 

In total, 32 cannabis samples were analyzed including hashish, leaves and flower buds. The team utilized methanol, ethanol and acetonitrile, and several combinations of the three, for cannabinoid extraction. After evaluating their results, they identified 80% acetonitrile mixed with 20% methanol as the best extraction solvent system. Utilizing this approach, they achieved 97-105% recovery of analytes of interest – a significant improvement on established methods. They combined UHPLC with photodiode array and single quadrupole mass spectrometry detectors (UHPLC-UV-MS) and tested several chromatography columns to identify the optimal set up. The authors state “The best separation and peak shape were achieved on a 100mm x 2.1mm Cortecs UPLC C18 Column”. They also found that a solvent composition of acetonitrile with 0.05% formic acid and 0.05% formic acid in water as the mobile phase enabled optimal chromatographic separation. Results were found to be highly reproducible with a low standard error. As such, the method was deemed to be suitable for routine cannabinoid analysis. 

Enhancing all aspects of cannabis analysis 

In a related Planta Medica paper, the same team recently published results on the detection and quantification of cannabinoids in extracts of C. sativa roots. They believe this is the first report to look at the quantification of cannabinoids in cannabis roots, highlighting their leadership in the field. In addition, Dr ElSohly mentioned that his team are working on the development of a method for the detection and quantitation of pesticides in cannabis. Putting the group’s work and focus on cannabis analysis in to broader context, Dr ElSohly ended by saying “One of our core aims is to detect any trends in the potency of confiscated materials, the introduction of any new varieties with unusual cannabinoid content, and enhance our ability to detect and quantify common cannabis contaminants. We publish extensively in these areas and will continue to do so.”