In the context of efforts to develop consensus-based reference methods for cannabinoid analysis, this tutorial discusses the methods to prepare samples for analysis in consensus methods. Specifically, the steps behind the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method of dispersive solid-phase extraction (dSPE) and salting out are explained as applied to the analysis of cannabinoids of interest.
Precise measurements are vital to any transaction of commerce. As the medical marijuana industry grows in the United States, in addition to standard weights and monetary measures, potency, impurities, and related measures become important. These measures are exacerbated by two external factors. First, the industry has its roots in the counterculture movement and the associated resistance to (governmental) regulation. Second, and more fundamental, since cannabis is illegal under federal law, individual states are developing their regulations on a state-by-state basis.
In addition to measuring purity, potency determination is another key factor to ensure that the dose is consistent between different forms of cannabis product. Until the industry is regulated on a more widespread level, it would not be unexpected for cannabis products to have a wide range of cannabinoid levels. Additionally, potency can vary with the stage of the growing season, the part of the plant harvested, or different plant strains.
To bring standardization to cannabis testing, especially in anticipation of future federal legalization and regulation, AOAC International has led the way—along with Spex CertiPrep and Spex Sample Prep, Sigma-Aldrich, GW Pharmaceuticals, Sciex, CEM Corporation, and SC Labs—through its working groups initiative. A draft fitness-for-purpose statement and standard method performance requirements are being considered. These will define the minimum performance characteristics necessary to evaluate quantitative analytical methods. Ultimately, quantitative methods are needed for measurements of cannabinoids in raw materials and formulations like topical creams and foods. Plans are for the required determination of five cannabinoids (see Table I) with an additional five cannabinoids listed as desirable (cannabichromene, CAS No. 20675-51-8; cannabichromenic acid, CAS No. 20408-52-0; cannabidivarinic acid, CAS No. 31932-13-5; cannabigerol, CAS No. 25654-31-3; cannabigerolic acid, CAS No. 25555-57-1; cannabidivarin, CAS No. 24274-48-4; Δ8-tetrahydrocannabinol, CAS No. 5957-75-5; tetrahydrocannabivarin, CAS No. 28172-17-0; and tetrahydrocannabivarin acid, CAS No. 28172-17-0). (See upper right for Table I, click to enlarge.) While Cerilliant has standards available for all five of the required cannabinoids, Restek, Sigma-Aldrich, API Standards, Echo Pharmaceutical, Lipomed AG, and the United States Pharmacopeia (USP) each have standards available for one or more of the cannabinoids required or desired for testing. Further information on the involvement of AOAC International with cannabis testing can be found on their website, www.aoac.org.
Surprisingly, there is little published literature on the extraction of cannabis or cannabis products for the isolation of cannabinoids before analysis. However, it seems that those involved in this testing are settling on the technique known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Developed in 2003 by Anastassiades and Lehotay (1) for the determination of pesticides in fruits and vegetables, the utility of the technique has been extended to a multitude of residues or natural products in plant matter, meats, soil, and other complex sample matrices. QuEChERS combines the well-established analytical approaches of salting out followed by dispersive solid-phase extraction (dSPE). This general scheme is outlined in Figure 1. (See upper right for Figure 1, click to enlarge.) Some vendors, such as those involved with the AOAC working group, are actively involved with developing QuEChERS for the extraction of cannabinoids from cannabis products and can be considered good partners for those laboratories involved with such determinations. To isolate cannabinoids from cannabis products for subsequent analysis, these general steps are followed:
Before GC analysis, the acid cannabinoids, cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA), must be derivatized by silylation to enhance analyte volatility and prevent undesirable adsorption on the column. Typically, MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) is used.
As the medical marijuana industry continues to gain acceptance and recognition, standardization of products and analytical methods for purity testing will move to the forefront. This will be driven by efforts such as those led by the AOAC working group and the QuEChERS extraction approach is very likely the method of choice for these determinations.
Douglas E. Raynie is with the Department of Chemistry and Biochemistry at South Dakota State University in Brookings, South Dakota. Direct correspondence to: Douglas.Raynie@sdstate.edu
D.E. Raynie, Cannabis Science and Technology 1(1), 58-60 (2018).