Advanced Cannabinoid Analysis - Latest Method Developments

Cannabinoids are a diverse group diterpenoid compounds primarily observed in Cannabis and Rhododendron species. To date, over 120 phytocannabinoids have been identified and quantified in Cannabis extracts using analytical techniques such as High Performance Liquid Chromatography (HPLC). With the federal legalization of hemp, a type of Cannabis, and state-supported legalization measures for high-THC Cannabis, HPLC testing of dried plant material for psychotropic potency and therapeutic dosing has become part of nearly every piece of legislation. While numerous chromatographic methods have been developed for the detection and quantification of THCA, CBDA, CBGA, CBNA, and their decarboxylated forms, many do not account for the possibility of coelutions with other secondary metabolites in plant samples such as cannabinoids, flavonoids, and terpenes. To complicate analyses further, the metabolomes of different Cannabis varieties can vary greatly, resulting in chromatographic coelutions that are present in some extracts but not in others.

The method presented in this application note attempts to resolve most of the significant coelutions common to different types of Cannabis and was designed for laboratories interested in the quantification of minor cannabinoid and cannflavin constituents. Using this method, a total of 34 unique Cannabis analytes were quantified in less than 32 minutes. The method described has been successfully applied to not only leaf and flower Cannabis tissue, but cannabis/hemp products such as concentrates, oils, and cosmetic products.

The cannabis market has been rapidly growing with the list of identified cannabinoids constantly increasing. Analytical methods for detecting over 25 cannabinoids by means of HPLC (High Pressure Liquid Chromatography) analysis have been explored and currently there are methods to separate 21 cannabinoids in a single run. Expanding the list of cannabinoids is possible, however stereoisomers challenge the current methods and, therefore, further method optimization is needed. Shimadzu’s new analytical method development software, LabSolutions MD (Method Development) alleviates the tedious task of testing, analyzing, and comparing all the individual runs. LabSolutions MD uses “Analytical Quality by Design (AQbD)” concepts to determine the optimal method for cannabinoid separation. Experimental design during the entire method development process, identification of the most robust analytical conditions, and predicted of chromatograms, provide the user with the power to automate the development of robust analytical methods. This software can be an asset to many fields and will be used in this study to efficiently separate cannabis stereoisomers.

With the passing of the Farm Bill in December 2018, which legalized hemp if the psychoactive compound, THC, content is 0.3% or less, and the legalization of cannabis in more than two thirds of US states and Canada, more accurate and sensitive analytical methods are needed for the quantitative determination of cannabinoids besides the widely applied technique of HPLC with UV and photodiode array detectors. The higher sensitivity, specificity, and mass identification provided by LC-MS are increasingly recognized for the quantitative determination of cannabinoids. In a previous application note (HPLC-028), an in-source fragmentation (SID) method was developed using the Shimadzu single quadrupole MS (LCMS-2020) to characterize 16 cannabinoids.

In this extended study, an LC-MS method of quantitation and simultaneous SID was developed using the LCMS-2020 single quadrupole MS with an integrated LC front end (LC 2040C 3D) for the quantification of 16 cannabinoids, including ∆9- tetrahydrocannabinol (∆9-THC), ∆8- tetrahydrocannabinol (∆8-THC), cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabicyclol (CBL) and their respective acidic forms, etc. Selected ion monitoring (SIM) was used for quantitation, simultaneous positive and negative scans were used for identification.