Pre-analytics is Catching Up to Cannabis Analytics at CSW 2019

Pre-analytics is Catching Up to Cannabis Analytics at CSW 2019

Cannabis Science West 2019 attracted almost 4000 scientists to the Portland OR, Convention Center on Sept 4-6 to talk shop about cannabis science in breeding, growing, harvesting and processing cannabis plants. The three parallel tracks were growing, analytical, and medicinal. I chose to focus on the analytical track.

The lectures reported interesting advances in the analytics, for example, one report found more than 1000 small molecules using ultrahigh-resolution mass spectrometry of cannabis plant material. Simply identifying them is a major project. But, what about quantitative analysis? One needs reference and working standards. Some exist, but there are still problems.

The boot camp on September 5, took the audience to a hemp grow in West Portland. Four immaculate, 10-acre open plots were allocated to different cultivars. The intended end use of the entire grow was to produce CBD extract for medicinal purposes from hemp cultivars. The owner of the grow pointed out that that the growth cycle of the cultivars was different in detail. One needed to know the plant and pay attention to its idiosyncrasies to avoid harvesting before or after its prime. Plus, the parts of the plant are important. CBD content of the flowers at the base are much generally much lower than at the top. Leaves have lower content than flowers and trichomes. Stems are lower still, but they have weight, and most sales are on a weight basis. So, what do you harvest, and how? With machine? By hand? If, by hand, how do you pay each harvester?

Then there are the regulations. Hemp cannot have more than 0.3% THC + THCA, where the A is for the acid, which decarboxylates to THC, the psychoactive component. Again, this seems to be an opportunity for blending to keep within the legal limits. Infused or spiked cannabis products are still another issue. Staff handling plant product early in the supply chain may infuse THC oil or mix hot THC plants suffering from high metal, pesticide or microbiology problems to pass contamination limits.

Over the last two years, many labs have formed and are starting to deliver results, including certificates of analysis, (CoA)s which are being used to support and facilitate commerce, supply chain management and local regulation particularly in the USA. Consensus on analytical methods is appearing, which is validated in operating laboratories. The need for comparability and integrity is gaining acceptance by growers, processors, and retailers. Buyers along the supply chain are comparing results from their labs with the CoA submitted by the sellers. Anecdotal reports of the comparisons focus on discrepancies that are hard to explain, except by fraud.  Integrity is suffering. In the analytical track, one lab manager repeatedly asked how significant are inter-laboratory differences? What is the reason why the CoA differs from the incoming inspection by 20% or more? The question is legitimate, but the few responses were long, complicated and not convincing. The NAS report this summer deals with improving repeatability and replicability in laboratory results1.

The scientific basis of the analysis of cannabis products in 2019 is filled with gaping holes and inconsistencies. All too many examples show linearity by serial dilution. How do real samples behave? The lectures and exposition at the meeting show a large improvement in the analytics-driven tools for the assays of cannabis as there is a progression toward consensus. The larger instrument vendors, including Advion, Agilent, Bruker, PerkinElmer, Sciex, Thermo, and Waters all can show replicability (%CV) less than 5% and often less than 2% for the analytics stage. Today, the pre-analytics stage, which includes sample collection from the plant, drying, storage, and sample prep has several weak links in the assay chain.

I left the Bootcamp wondering, with so many variables, nearly all of which are uncontrolled, how long will it take to develop a useful and rigorous scientific base for cannabis products? Our society needs much more science to support science-based regulation. Current practice, which is legislatively driven, is inconsistent between the numerous jurisdictions. Worse, the regulations are not consistent with the idiosyncrasies of the plant or product. For example, Oregon requires that hemp, for medicinal CBD, be harvested within 28 days of the date of analysis of the plant material for THC content, which must be less than 0.3% dry weight.

The grower must predict the harvest date before submitting samples for THC analysis. However, the cultivars have different growing cycles. Weather in Oregon is notoriously variable in late August through mid-October, which is the harvest season for most hemp cultivars. The distribution of THC in the cannabis plants also is an uncontrolled variable.  Fresh flowers at the top may have much more THC in the trichomes than the flowers near the base of the plant. This raises the prospect of data-driven selective sampling in the field, prior to sending the hemp sample to the reference lab to get a Certificate of Analysis of less than 0.3% THC.

Terpenes were another important topic, especially for edibles and smokables, since they are volatile and responsible for much of the flavor. Fingerprint chromatography of Cannabis Sativa of cannabis can produce complex chromatograms with several hundred peaks, many of which are partially overlapping. Different strains have different profiles. Plus, the profiles change with plant maturity and location in the plant. Method of harvest and drying conditions are other variables that cry out for scientific study and control.

Pre-analytics

The most noteworthy new products featured at Cannabis Science West lectures, posters or exposition were focused on the pre-analytics stage.

Novel Sampling Techniques from Advion

The Advion (Ithaca, NY) booth, which was just inside the exhibition entrance, featured the automobile portable Expression Compact Mass Spectrometer (CMS) which Professor Jack Henion, Advion’s founder and now VP of research, described in his lecture in the analytical track. Advion introduced the ASAP Direct Sample Probe which provides samples for direct sampling and injection of cannabis plant material in 30 seconds. The ASAP employs a disposable glass capillary melting point tube to assure no carry-over issues when running back-to-back samples. No solvent or chromatography is required.

To compete with other atmospheric pressure ionization sources, Advion introduced the Touch Express Open Port Sampling Interface (OPSI) as a general-purpose sampling technique using electrospray ionization. Picture a small meta bloc with a vertical hole heading down toward the ESI orifice. A side hole delivers a flowing liquid to the top of the ESI hole, where a meniscus is formed. The venturi created by the ESI source sucks the liquid from the meniscus down to the ESI orifice. The liquid meniscus is open to the air, thus sampling it nearly instantaneously. Or one can touch Fibers such as SPME to the liquid, which washes analytes off the fiber and into the ESI and into the MS.

Both of these devices simplify sample processing while providing immediate results that can be used in crop management at-site. Plus, if one adds a gradient elution liquid chromatograph to the CMS, one can assay for pesticide residues. Given the value of cannabis crops, the instruments can quickly pay for themselves. However, the MS itself may require an experienced operator.

Aokin Mycontrol Analyzer FP

The value of cannabis plant products is high enough to support manual operations such as walking the fields to inspect plants for health and gender. Males and hermaphrodite cannabis plants are undesirable and culled. Field hands also look for signs of microbial infection, which can ruin the plant for its intended use.

Aokin (Berlin, Germany) introduced the mycontrol analyzer for rapid quantitative analysis of mycotoxins, herbicide residues, THC and other analytes. The mycontrol uses fluorescence polarization assays to read liquid from a solid-phase extraction cartridge. The process is simple (weigh, extract, filter, dilute and analyze). The fluorescence photometer is small enough for at-plant analysis. The RSD for the THC kit is +/- 4%, with a LoD of 100 mg/kg. For the aokin mycontrol kit, the RSD is +/- 5% with a detection limit of 0.1 ug/kg. These CVs compare favorably with lab results using HPLC. Aokin has developed assay kits for total aflatoxins, aflatoxin M1, fumonisin, total THC and more.

Infused Products

Over half of the Cannabis market involves cannabis for smoking, even without vaping. Deaths and lung damage from vaping was in the popular news while the conference was in session. Infusing cannabis with other volatile materials including fentanyl was suspected. However, this was often conjecture, since the etiology of the lung problems was, and still is, not clear. 

To make sense of the lung problems, one needs information on the chemicals infused into the cannabis products. Entech Instruments (Simi Valley, CA) introduced Vacuum Assisted Sorbent Extraction (VASE) platform as the first stage of a thermal desorption GC-MS to profile cannabis-infused cannabis products. 

The VASE Sorbent Pens are plug-in application-specific modules. The all Glass Sorbent Pen (GSP) provides direct thermal desorption from the sample. It is used to sample cannabis for terpenes, cannabinoids and infused chemicals. In addition to infused smokable, it is often used for gummies, flavorings such as vanillin, and residual solvents. The Headspace Sorbent Pen is designed to sample the headspace over a liquid in a vial. The diffusive sorbent Pen and Active Sorbent Pen are used for personal monitoring of analytes. For desorption, the pens are placed in the TD desorption chamber. When the chamber is closed, hot carrier gas desorbs the analytes directly into the Capillary GC column. Infused cannabis GC products normally use the Glass Sorbent Pen.

Photonion GmbH (Schwerin, Germany) introduced a new smoke analyzer for characterization of cannabis smoke. The instrument focuses vacuum UV light (10.78 eV) on the smoke aerosol which has the energy to ionize many compounds which are analyzed by an integrated ToF MS. The MS has a range of 1 to 600 AMU. Mass resolution is 700. Dynamic range is 10^6. Detection limit is 100 ppb of benzene. The performance of the instrument was discussed during a lecture by Prof Ralf Zimmerman of the University of Rostock, Germany.

There is a huge history of studies on tobacco smoke. It will be interesting to see how tobacco smoke compares with cannabis and infused plant materials. Please see the recent posting on INTERVAL from PMI2

Interesting New Technology

CEM optimizes SMART Q™ for rapid water assay of cannabis plant material. Water content of cannabis plant material is important since some measurements are specified on a dry weight basis. Plus, high water content can support the growth of microbes, leading to toxic residues. CEM introduced the SMART Q ™ infrared water analyzer. The instrument features a novel optical design that measures the temperature of the entire sample, rather than just a hot spot.  Application examples included hemp flower, CBD extract, gummy candy, compared to air oven technology, SMART Q runs were completed in less than five minutes, compared to multi hours required for air oven assays. A comparison of the average % difference between the two methods was 0.07%, which is an excellent agreement between two orthogonal methods.

Reference Standards from LGC

Cannabis standards are required for almost all quantitative assays of cannabis products. LGC (formerly Laboratory of the Government Chemist in the UK) introduced the Dr. Ehrenstorfer ™ line of reference materials in solution at 1000 ug/mL concentration. Currently available standards include Cannabicyclol (CBL), (-) – CBD, Cannabidiiolic acid (CBDA), Cannabinol (CBN). (-)-D 8 THC, (-)-D 9-THC and D 9-THC.

A lecture by Michael Hurst of Millipore’s Reference Materials team presented disturbing evidence of accelerated degradation of cannabis standards when mixed with others. Pure reference standards in solution are stable to degradation for months. However, when the ampule was opened and mixed with other standards to simulate a mixture of cannabinoids, degradation was noticed within a few days. The cause is not known, but analysts should be warned that reference standards of cannabinoids may have a short life. Possible causes include oxygen and/or light exposure. 

Stability of Cannabinoid Oils with Metrohm’s Rancimat

Oils turn rancid upon storage due to slow oxidation. Cannabis oils produced by distillation or extraction are also prone to rancidity, which decreases their value for edibles. A poster from Stillwater Brands (Commerce City, CO) and Metrohm (Riverview FL) showed that cannabinoid oils are generally much more stable than common food oils. High D 9-THC oils are less stable when other cannabinoids are present. Repeated heat cycling of high D9-THC oils also reduces oil stability.

Purification of Cannabis

Distillation of cannabis products, especially oils is a common process. Arometrix, Inc. (Rockland, MD) introduced the Fraction Finder for monitoring the distillate on laboratory scale (~ one liter) and larger process scale distillation. The Fraction Finder uses fluorescence signal generated by a fluorescent ring at the exit of the condenser in a conventional distillation glass apparatus. This novel instrument helps improve product consistency by measuring the “heads”, “bodies” and “tales” so the cut points are consistent. This aides in hitting the sweet spot for the specifications for downstream processing.

Cannabis plant material is heterogeneous, which complicates taking a representative sample for analysis. This was discussed during a session at the Boot Camp organized by Retch ® (Haan, Germany). Retsch produces a wide range of mills supported by milling protocols covering the range of liquid nitrogen frozen to elevated temperature. The GM 200 was featured as new. It has a 1000-watt motor which powers the rotating blade at 14,000 RPM. The high speed facilitates the grinding of fibrous plants such as hemp and Cannabis Sativa in seconds with sample volume up to 700 mL of plant material. The lid is gravity loaded, so it settles as the sample volume is reduced by milling.

Chromatography of Cannabis

Restek was one of the early developers of LC and GC chromatography columns and supporting analytical standards.  They prepared a concise product summary for cannabis applications. One LC Chromatogram showed the baseline separation of 16 cannabinoids in less than 9 minutes using a Raptor ARC-18 column packed with 2.7 um particles. The next page showed a separation of 10 critical cannabinoids in less than 200 seconds using a Rxi GC capillary column. The next example focused on terpene profiling, again with GC with 36 analytes in 17 minutes. Restek points out that they help build workflows that use the same columns and consumables. They are sensitive to the cost of the many assays, and hence potential startup inventory, required by assays of cannabis products.

Thin Layer Chromatography for Scanning Samples

Sorbtech introduced the TLC Rocket Development Chamber. Thin Layer Chromatography is suitable for qualitative analysis of cannabis materials when a quick check is sufficient. Sorbtech’s (Norcross GA) TLC Rocket is a new design that uses a limited amount of solvent to automatically stop the run when the solvent is depleted. This facilitates “fire and forget” operation. Solvent consumption is reduced by 80 – 85%, with much more comparable results. Sorbtech also introduced a range of cartridge columns for flash chromatography.

Summary: The science base supporting the cannabis renaissance is growing to meet demand for safety, efficacy, and consistency. Stakeholders in the industry providing cannabis commerce need to critically review the supply chain to make sure the consuming public is protected. Remember, there is a black market that is competing on price to supply the consumer. Any scandal in the licit supply chain will decrease the value of the science and products we are trying to quickly develop.

Credits

The fourth Cannabis Science Conference West (2019) was organized by CSC Events, LLC to mark the year-to-year improvement in the science related to cannabis. Josh Crossney deserves special recognition for serving as chief visionary, President and CEO of CSC and CSC events. The global society needs to significantly improve the science base of cannabis. This meeting continues to make a positive contribution to this endeavor.

References

1. National Academies of Sciences. Engineering, and Medicine. 2019. Reproducibility and Replicability in Science. Washington, DC: The National Academies Press. https://doi.org/10.17226/25303

2. Manuel, P.C., Boue, S. (2019, September 23) Facilitating Reproducibility in Data-Rich Sciences. Retrieved from https://www.labcompare.com/10-Featured-Articles/517908-Facilitating-Reproducibility-in-Data-Rich-Sciences/

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