Medicinal Cannabis and the Need for Enhanced Cannabinoid ProfilingKen Kovash1, Melodie Kovash1, Paul Winkler2, Ken Tanaka2, David Yazdi2, Will Bankert2, Bob Clifford2, William Lipps2 and Scott Kuzdzal2

1G.I. Grow, Deer Island, Oregon. 2Shimadzu Scientific Instruments, Columbia, Maryland.

Melly Nova G.I.

Medicinal cannabis refers to the use of cannabis and its corresponding cannabinoids, as a therapy to treatdiseases and/or alleviate symptoms. While less than 6% of today’s studies on marijuana analyze its medicalproperties, publications to date indicate that cannabis shows great promise for the treatment of manydiseases and symptoms. The cannabis industry is projected to be an $8B industry by 2018. With this growthhas come an explosion in cannabis testing labs, which perform testing that ensures patients receive safeproducts free from pesticides, contaminants, residual solvents and microorganisms.

The premium products sold in medicinal cannabisdispensaries are typically those with high THCconcentrations, however, it’s THC-A and the othernon-psychoactive “CBx” cannabinoids that have beenreported to reduce convulsions, inflammation, nauseaand anxiety, and even eradicate tumors in somepatients2. CBN, only slightly psychoactive, is adegradation byproduct of THC and it is elevated whencannabis is poorly stored.

HPLC, LCMS and GCMS enable accurate cannabis testing (including potency, terpenes, pesticides, residual solvents, etc.).Although less than ten cannabinoids are routinely measured in cannabis product quality control, cannabis plants containhundreds of unique compounds. As use of medicinal cannabis increases, testing must expand beyond the a handful ofcommonly measured cannabinoids (and terpenoids) to larger CBx profiles. This increased testing will help us learn moreabout health benefits, and develop more personalized cannabinomics strategies. We must also better further ourunderstanding of cannabinoid/terpenoid/flavonoid synergies. We are currently investigating ion trap time-of-flight MS,immuno-MS and SFE-SFC-MS in attempt to characterize a more comprehensive cannabinome. We are also applying MALDI-TOF MS for seed and dry product strain typing. More research is needed to further our understanding ofcannabinoid/terpenoid/flavonoid synergies.

① INTRODUCTION

② CANNABINOMICS

③ METHODS

④ POTENCY PROFILING

⑤ PERSONALIZED CANNABINOMICS

⑥ SUMMARY

Cannabinoid standards (Cerilliant and Cayman) were adjusted to 50mg/L.Potency profiling was performed using the Shimadzu Nexera-i PDA (LC-2040C3D). Mobile phases A: 0.1% formic acid in water, B: 0.1% formic acid in ACN,column: RESTEK Raptor ARC-18 2.7um, 150 x 3.0mm (this column enablessub 2-minute runs without the need for UHPLC), flow rate: 2.0ml/min,column temp.: 50C, injection volume: 1uL. Cannabinoids fromcannaceuticals (G.I. Grow) were extracted from natural products withmethanol or IPA and analyzed in Oregon.Terpene profiling and residual solvent analyses were performed using aShimadzu GCMS Ultra with HS-20 headspace. Pesticides were also analyzedto certify products as contamination-free (but omitted from this poster dueto spatial restraints). Contact us for details.

Nexera-i integratedHPLC System.

GCMS Ultra with HS-20. This instrument has twin-line capabilities for simultaneous terpene/residual solvent testing.

Twenty-three U.S. states (plus D.C.) have legalized medicinal marijuana.Four states have legalized recreational use. It is estimated that 18 additional states are considering legalizing recreational cannabis.

Medical marijuana dispensaries offer many forms of cannabis products, ranging from dry products and concentrated oils to edibles and beverages. Medical marijuana card holders typically pay less than recreational cannabis users. Cannabis consumers select their own ‘cannaceuticals’, but websites like Leafly.com have emerged to provide information and assist consumers with purchases.

G.I. Grow strictly abides by Oregon Medical Marijuana Program rules and ispioneering a personalized cannaceutical approach. G.I. Grow nurtures cannabisplants, focusing on quality rather than yield, to produce blends of CBx oils inresponse to patient outcomes, tailoring personalized cannabinomics strategiesfor each individual patient. Cannabis produces different chemicals due tomultiple environmental/physical stresses and needs. The figures to the rightshow G.I. Grow’s natural sunlight farm, as well as several G.I. Growcannaceuticals (right). Cannabinomic analyses provide a better insight andunderstanding of the potential health benefits of cannabis. The cannabinoid‘heat map’ (right) shows formulation data for many G.I. Grow cannaceuticals.

Cannabis contains more than

480 compounds that are unique to cannabis, including

86 known cannabinoids.

36

The marijuana plant contains several chemicals that mayprove useful for treating a range of illnesses or symptoms,leading many people to argue that it should be made legallyavailable for medical purposes1.

Tetrahydrocannabinol (THC) does not occur naturally incannabis. Tetrahydrocannabinolic acid (THC-A) is the natural,non-psychoactive, carboxylic acid form of THC. It is rapidlyconverted to THC upon smoking or heating. Other naturalcannabinoids, such as cannabidiol (CBD), are reported to havemany potential health benefits, including anti-inflammatory anti-anxiety, anti-epileptic, sedative, and neuro-protectiveproperties2. Unfortunately, most marijuana sold to consumershas reduced levels of CBD because many growers selectivelybreed out the CBD enzyme to produce more THC.

1NIH Website entitled, “DrugFacts: Is Marijuana Medicine?” http://www.drugabuse.gov/publications/drugfacts/marijuana-medicine2Potential health benefits were collected from 36 peer reviewed journals and news articles. For summaries, see “23 Health Benefits of Marijuana” from Business Insider (April 20, 2014) and Marijuana and Medicine: Assessing the Science Base, Joy, Watson and Benson, National Academy Press, DC, 1999, Chapter 4: The Medical Value of Marijuana and Related Substances.

For Research Use Only. Not for Use in Diagnostic Procedures.

One cannabis oil success story is that of Elkan, now 10 years old, living inOregon. Elkan suffered from severe autism, including Attention DeficitDisorder, Attention Deficit Hyperactivity Disorder, PervasiveDevelopmental Disorder and Sensory Processing Disorder. Elkan also hadtrouble speaking, suffered intense leaky gut syndrome symptoms andneeded to be physically restrained. Elkan’s mother, Laura commentedthat, “Elkan’s doctors were not sure why all pharmaceuticals other thanRitalin were showing no benefit whatsoever.” Nothing seemed to workand most pharmaceuticals only exacerbated his symptoms. Elkan begantaking a blend of natural cannabis oils and within three months ofpersonalized cannabis treatment, he can now speak, does notexperience Leaky Gut Syndrome symptoms and no longer needs to berestrained.

HPLC and GCMS analyses of Elkan’s personalized cannaceutical show ahigh THC-A content, as well as a unique, customized terpene profile(right). Cannabinoids receive much focus, but it is the synergy betweencannabinoids and terpenoids (and other compounds such as flavonoids)that delivers maximum therapeutic effect. For example, the terpenepinene, which gives cannabis its pine odor, acts as a bronchodilator,opening the lungs and increases cannabinoid absorption (as in thebronchodilation you may experience when hiking in a pine forest). Thisalso makes high pinene concentrations beneficial for COPD and asthma.Linalool increases sedation, but makes consumers drool. A betterunderstanding of these complex synergies and effects is needed.

Analytical testing is necessary to ensure that consumers receive accurately labeled products that are free fromcontamination. Today’s cannabis has significantly higher THC-A levels. HPLC analysis of a potent strain from G.I. Grow, anorganic, biomedical farm in Oregon, shows a Δ9 THC-A potency of 31.8% (below, left). HPLC cannabis methods arecontinuously improving. A 2-minute separation of all major cannabinoids is presented below (right).

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Peaks1. Cannabidiolic acid (CBDA)2. Cannabigerolic acid (CBGA)3. Cannabigerol (CBG)4. Cannabidiol (CBD)5. Tetrahydrocannabivarin (THCV)6. Cannabinol (CBN)7. Δ-9-Tetrahydrocannabinol (Δ9-THC)8. Cannabichromene (CBC)9. Δ-9-Tetrahydrocannabinolic acid (THCA)