All analytical sciences rely on reference standards—standardized, punctiliously prepared and characterized samples—to guarantee the veracity of their results but even more importantly, of their methodology. Cannabis is a complex botanical capable of generating numerous downstream products, each with unique critical quality attributes. And, as a botanical, it is itself an extremely complex matrix.
“My impression is that most people use the terms ‘reference standard’ and ‘reference material’ interchangeably, but they’re not the same thing,” says Dr. Sid Sudberg, founder of Alkemist Labs (Garden Grove, CA) and technical advisor for the American Herbal Pharmacopoeia.
According to Sudberg, a reference material is a substance, botanical, or a multi-component preparation from the plant, whereas reference standards are single chemical compounds or pure substances of known concentrations, which may be directly compared with values from samples after constructing an appropriate concentration graph. “The taxonomically identified plant in its flowering state is considered the gold standard of botanical identification and will nearly always suffice as a valid reference material for botanicals,” Sudberg adds. “Since flowering plants are not always available, we rely on several different levels of authentication.”
Variations on the authentication process commonly used to create valid botanical reference materials require identification methods with reasonably high specificity, along with a reasonable number of closely-related species and/or plant parts, so as to rule out a pure specimen from a closely related species.
“Botanical reference materials are created by bringing all available characterization resources to bear on qualified botanical materials. Considerations include the source and documentation provided to ensure sample authenticity, and evidence for chain of custody,” Sudberg says. “The analytical methodology, plus documentation supporting authenticity, should be robust and reproducible.”
In some instances, a component or phytochemical unique to the reference material or species under investigation may be used to identify plants. For cannabis, that compound would be THC since it is found nowhere else in nature. Sudberg cautions against carrying this idea too far, however. “Most botanicals lack specific compounds that clearly distinguish them from other species. Phytochemicals are quite ubiquitous and cannot be used to make a definitive identification.”
A laboratory’s preferred analytic methodology and instrumentation always factors in decisionmaking on reference standards and materials. “Our motto has always been, ‘the more data, the better, the more confident we can be with any botanical identification,” Sudberg says. “Ultimately, many different methodologies might be useful but ideally these methodologies should be used together, orthogonally, to increase the level of confidence with any given authentication.”
Reference standards versus reference materials
“Many standards exist for botanical products,” says Patricia Atkins, senior application scientist at SPEX CertiPrep (Metuchen, NJ). “Some reference standards are for particular analytes like heavy metals or pesticides, which are prepared at specific concentration in a designated solvent.” Reference materials and standards that match the sample’s matrix may be an authentic reference material with known or designated amounts of, for example, pesticide residues. “Depending on the endpoint, different standards may be needed or preferred.”
An ideal matrix-matched standard for cannabis might consist of a cannabis plant standardized for the different analytical targets such as terpenes, heavy metals, pesticides, psychoactive ingredients, etc.
Labs requiring traceable quantitative standards should be using certified reference materials (CRMs) as their primary standards. The international standard governing the manufacture of reference materials, ISO 17034:2016, mandates that the standards be metrologically traceable, and that manufacturers report certified concentrations with corresponding measurement uncertainties.
“Traceability assures end users that the reported measurement agrees with results obtained from other laboratories using the same standard,” says Roxanne Franckowski, manager of ISO Quality at Cayman Chemical (Ann Arbor, MI). “Note that certified reference materials are only as good as the laboratory equipment used to analyze them, so proper instrument calibration is key for achieving traceability.”
Cayman tests its CRMs under ISO/IEC 17025:2017 to establish metrological traceability, which ISO/IEC Guide 99 defines in terms of a reference through “a documented, unbroken chain of calibrations, each contributing to measurement uncertainty.”
“Single analyte CRMs have been the default option for analytical testing,” Franckowski says. “Multi-component CRMs are now emerging, which provide several advantages.” Single-component CRMs allow labs to create their own multi-component control samples at varying concentrations, while a multi-component CRM may be ideal when creating calibration standards. “Using commercially formulated multi-component CRMs can help labs reduce dilution errors, material consumption, and preparation time, and provide an opportunity to create custom, multi-component standards that fit specific testing requirements,” explains Franckowski.
Accuracy is paramount
According to Morgan Fox, a policy advocate with the National Cannabis Industry Association (Washington, DC), the status of cannabis as a Schedule 1 drug under the US Controlled Substances Act is the primary barrier to establishing standards and uniform protocols in cannabis testing. “Since cannabis is federally illegal, it is extremely difficult to get permission to study it or obtain relevant research materials. It also prevents federal agencies from regulating cannabis or issuing guidance around testing.” Current law forbids even the transportation of THC-containing cannabis products between US states. That leaves regulating cannabis testing to local jurisdictions which, Fox says, have done “an excellent job” of keeping potentially impure or dangerous products off the market. “However, until we change federal law, we will be unable to implement best practices and standards across the board, and research will continue to be stymied.”
Regulatory hurdles to using cannabis reference materials depend on a lab’s status. Because it is registered with the US Drug Enforcement Agency, Alkemist is relatively unencumbered. “The only regulatory hurdle I can think of for us is having in hand cannabis materials with sufficiently high THC content,” says Sudberg.
That being said, accuracy (trustworthiness) is paramount. “That is why we always try to do as much as possible to increase our level of confidence in making correct botanical identifications, which will vary from one botanical to another,” Sudberg says. “We decide case-by-case what suffices to make an accurate identification, based on the needs of any given analysis. Possessing the knowledge and experience to make this determination is why the selection of a testing lab is not to be done without careful vetting.”