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Publication Title | Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis

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Giese et al.: Journal of AOAC International Vol. 98, No. 6, 2015 1503 DRUG FORMULATIONS AND CLINICAL METHODS
Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis
Matthew W. Giese, Mark A. Lewis, Laura Giese, and Kevin M. Smith
Napro Research, Westlake Village, CA 91362
The requirements for an acceptable cannabis assay have changed dramatically over the years resulting in a large number of laboratories using a diverse array of analytical methodologies that have not been properly validated. Due to the lack of sufficiently validated methods, we conducted a single- laboratory validation study for the determination of cannabinoids and terpenes in a variety of commonly occurring cultivars. The procedure involves high- throughput homogenization to prepare sample extract, which is then profiled for cannabinoids and terpenes by HPLC-diode array detector and GC-flame ionization detector, respectively. Spike recovery studies for terpenes in the range of 0.03–1.5%
were carried out with analytical standards, while recovery studies for Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid, and cannabigerolic acid and their neutral counterparts in the range of 0.3–35% were carried out using cannabis extracts. In general, accuracy
at all levels was within 5%, and RSDs were less
than 3%. The interday and intraday repeatabilities
of the procedure were evaluated with five different cultivars of varying chemotype, again resulting in acceptable RSDs. As an example of the application of this assay, it was used to illustrate the variability seen in cannabis coming from very advanced indoor cultivation operations.
The requirements for an acceptable cannabis assay have changed dramatically over the years. Historically the focus was the quantification of Δ9-tetrahydrocannabinol (THC; 1), the main biologically active metabolite; however, intensive research over the past few decades has identified over 150 different cannabinoids (2). Pharmacological activities for a number of these, including Δ9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabivarinic acid (THCVA), cannabidivarin (CBDV), and cannabidivarinic acid (CBDVA; 3), have been reported. Thus, quantification of these other cannabinoids is also important to understanding the pharmacological properties of cannabis. As of 2014 there have
Received May 7, 2015. Accepted by AP July 20, 2015 Corresponding author’s e-mail: mgiese@naproresearch.com DOI: 10.5740/jaoacint.15-116
been over 545 constituents identified in cannabis (3), and apart from the cannabinoids, the 140 terpenoids (4) have garnered interest due to their organoleptic properties (4, 5), potential for chemically fingerprinting different cultivars (6–10), and putative synergistic interactions between the cannabinoids and the terpenoids (11, 12).
Of the 140 terpenoids identified in cannabis, our own qualitative GC/MS surveys of California landraces, as well as analytical results from other laboratories (13, 14), published studies on the terpene profiles of cannabis cultivars (7, 9, 10), and reviews of the biological activities of terpenes (11, 12) suggest there are approximately 17 that are the most common and can be used for examining the phenotypic and/or biological properties of cannabis cultivars. Publications documenting the chemotypic profiles of various cultivars (9, 10, 15–17), analytical results obtained within our laboratory, and analytical results posted by other cannabis testing laboratories (13, 14) also provide evidence that typical concentration ranges for the cannabinoids are from 0.1 to 40% of inflorescence dry weight and terpenoids range from 0.01 to 1.5% of inflorescence dry weight. Although there are certainly other classes of metabolites present in cannabis (2, 4), the above references as well as a survey of services offered by analytical laboratories that test cannabis (13, 14, 18) suggest much of the current focus lies with the cannabinoids and terpenoids. Thus, an economical, robust, and validated method for profiling all of these analytes over their observed ranges of concentration is needed.
As the number of states considering legalization of cannabis, medical or otherwise, is growing rapidly, the number of cannabis testing laboratories has also increased to keep up with demand; however, it is not always clear what standards they are held to, and this has resulted in a number of nonstandardized and nonvalidated methods being used (1, 19). While there may be a number of methods suitable for the analysis of cannabis, it is crucial for laboratories to perform rudimentary assay validation to demonstrate the assay is fit for its intended purpose. This is an absolute requirement for the confident use of any methodology, and the International Conference on Harmonization (ICH), United Nations Office on Drugs and Crime (UNDOC), and AOAC provide a number of guidance documents for this purpose (20–22). Based on the aforementioned considerations and the lack of a method that was validated for the analytes and concentration ranges of interest, we sought to develop an efficient and robust assay that covered the typical repertoire of analytes and working ranges mentioned above.
A number of publications have been released over the years (10, 15, 16, 23) that have presented validated assays for several analytes present in cannabis. These validations were suitable for the stated purposes of the studies; however, they

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