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ACS Fall 2022: Purple Rain

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ACS Fall 2022: Purple Rain

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Cannabidiol (CBD)-containing products have flooded the stores. With this new market segment, understanding of safety testing and regulatory oversight is still developing. We know that CBD is unstable in solution and some CBD products are anecdotally reported to turn purple on storage. HU-331, a readily-formed quinone derivative of CBD, is commonly identified in samples of CBD isolate and CBD-containing solutions such as e-liquids, and is frequently proposed as the origin for the purple discolouration of CBD-containing products.
We investigated its photo-induced degradation mechanism which leads to the discolouration in samples contaminated with HU-331, and isolate a previously unreported cannabinoid intermediate which we found rapidly reacts with oxygen to form a multitude of cannabinoid derivatives and reactive oxygen species. Notably, the identification of the novel intermediate resolves the unknown reactions pathways which lead to the discolouration of CBD-containing samples, and highlights the potentially destructive role that HU-331 contamination may cause. Our findings suggest that multiple unreported cannabinoid derivatives are likely present in aged CBD samples stored under ambient conditions with some likely responsible for the discolouration, calling for regulatory control of HU-331 contaminant levels in CBD products.

Cannabidiol (CBD)-containing products have flooded the stores. With this new market segment, understanding of safety testing and regulatory oversight is still developing. We know that CBD is unstable in solution and some CBD products are anecdotally reported to turn purple on storage. HU-331, a readily-formed quinone derivative of CBD, is commonly identified in samples of CBD isolate and CBD-containing solutions such as e-liquids, and is frequently proposed as the origin for the purple discolouration of CBD-containing products.
We investigated its photo-induced degradation mechanism which leads to the discolouration in samples contaminated with HU-331, and isolate a previously unreported cannabinoid intermediate which we found rapidly reacts with oxygen to form a multitude of cannabinoid derivatives and reactive oxygen species. Notably, the identification of the novel intermediate resolves the unknown reactions pathways which lead to the discolouration of CBD-containing samples, and highlights the potentially destructive role that HU-331 contamination may cause. Our findings suggest that multiple unreported cannabinoid derivatives are likely present in aged CBD samples stored under ambient conditions with some likely responsible for the discolouration, calling for regulatory control of HU-331 contaminant levels in CBD products.

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ACS Fall 2022: Purple Rain

  1. 1. Purple Rain: Discolouration Results from Cascading Photo- Redox Reactions Brodie Thomson, Dr. Markus Roggen
  2. 2. Introduction DELIC Labs is a research venture that seeks to add fundamental scientific insight to the field of cannabis and mushroom production. We seek to support the cannabis and mushroom industries by establishing a centralized hub in Vancouver, BC, for collaborative research focused on: • Process Design • Process Optimization • Process Analytics • Formulation Research
  3. 3. Collaborative Research DELIC Labs collaborates with academic, industry and private groups around the globe. Some highlights of those collaborations are: • University of British Columbia, Vancouver • Loyalist College, Belleville • Via Innovations by Dr. Monica Vialpando • Verdient Science by Dr. Linda Klumpers Fundamental Collaboration
  4. 4. Research Topics • Chemometrics and data analytics for process control and optimization • Kinetic studies to understand mechanisms • In-process analytics for process control • Computational studies to understand mechanisms • Process development, like crystallization Fundamental Cannabis and Mushroom Chemistry
  5. 5. 1 Outline - Degradation routes of CBD
  6. 6. 1 Outline - Degradation routes of CBD - Previous work: the search for HU-331’s anion
  7. 7. 1 Outline - Degradation routes of CBD - Previous work: the search for HU-331’s anion - Origin of orange and purple colours in CBD-containing solutions
  8. 8. CBD Usage [1] Dobian, CBD Market to reach $19.5 billion by 2025, but only with FDA approval says top researcher. Forbes. July 28, 2021. Available at https://www.forbes.com/sites/irisdorbian/?sh=2cd295c94bf8. 2 - Global CBD sales predicted to exceed US $19 billion by 2025.1
  9. 9. CBD Usage [1] Dobian, CBD Market to reach $19.5 billion by 2025, but only with FDA approval says top researcher. Forbes. July 28, 2021. Available at https://www.forbes.com/sites/irisdorbian/?sh=2cd295c94bf8. [2] Wheeler, M.; Williams Merten, J.; Gordon, B. T.; Hamadi, H. Substance Use & Misuse, 2020, 55, 1138-1145. 2 - Global CBD sales predicted to exceed US $19 billion by 2025.1 - Users of CBD (including E-liquids) are confused of both the current legality and regulation.2
  10. 10. CBD Usage [1] Dobian, CBD Market to reach $19.5 billion by 2025, but only with FDA approval says top researcher. Forbes. July 28, 2021. Available at https://www.forbes.com/sites/irisdorbian/?sh=2cd295c94bf8. [2] Wheeler, M.; Williams Merten, J.; Gordon, B. T.; Hamadi, H. Substance Use & Misuse, 2020, 55, 1138-1145. 2 - Global CBD sales predicted to exceed US $19 billion by 2025.1 - Users of CBD (including E-liquids) are confused of both the current legality and regulation.2 - Large number of CBD users report no framework for determining dosage.2
  11. 11. CBD Usage [1] Dobian, CBD Market to reach $19.5 billion by 2025, but only with FDA approval says top researcher. Forbes. July 28, 2021. Available at https://www.forbes.com/sites/irisdorbian/?sh=2cd295c94bf8. [2] Wheeler, M.; Williams Merten, J.; Gordon, B. T.; Hamadi, H. Substance Use & Misuse, 2020, 55, 1138-1145. [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. 2 - Global CBD sales predicted to exceed US $19 billion by 2025.1 - Users of CBD (including E-liquids) are confused of both the current legality and regulation.2 - Large number of CBD users report no framework for determining dosage.2 - Of 13 e-liquids tested, 38% of samples were labelled within 10% of reported CBD concentration.3
  12. 12. CBD Usage [1] Dobian, CBD Market to reach $19.5 billion by 2025, but only with FDA approval says top researcher. Forbes. July 28, 2021. Available at https://www.forbes.com/sites/irisdorbian/?sh=2cd295c94bf8. [2] Wheeler, M.; Williams Merten, J.; Gordon, B. T.; Hamadi, H. Substance Use & Misuse, 2020, 55, 1138-1145. [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. 2 -100 -80 -60 -40 -20 0 20 Deviation (%) Sample Tested Deviation from advertised CBD content - Global CBD sales predicted to exceed US $19 billion by 2025.1 - Users of CBD (including E-liquids) are confused of both the current legality and regulation.2 - Large number of CBD users report no framework for determining dosage.2 - Of 13 e-liquids tested, 38% of samples were labelled within 10% of reported CBD concentration.3
  13. 13. CBD Content Degradation 3
  14. 14. CBD Content Degradation - Average CBD degradation of 20% 30 days at 37°C.3 [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. 3 Heat
  15. 15. CBD Content Degradation - Average CBD degradation of 20% 30 days at 37°C.3 - Effect of light alone resulted in 15% CBD degradation in 30 days.3 [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. 3 Light Heat
  16. 16. CBD Content Degradation - Average CBD degradation of 20% 30 days at 37°C.3 - Effect of light alone resulted in 15% CBD degradation in 30 days.3 - Optimum CBD storage between pH 4-6.4 3 Light Heat Bronsted Acid [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. [4] Jaidee, W.; Siridechakorn, I.; Nessopa, S.; Wisuitiprot, V.; Chaiwangrach, N.; Ingkaninan, K.; Waranuch, N. Cannabis Cannabinoid Res. 2022, 7, 537-547.
  17. 17. CBD Content Degradation - Average CBD degradation of 20% 30 days at 37°C.3 - Effect of light alone resulted in 15% CBD degradation in 30 days.3 - Optimum CBD storage between pH 4-6.4 - Metal leaching – LA formation 3 Light Heat Bronsted Acid [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. [4] Jaidee, W.; Siridechakorn, I.; Nessopa, S.; Wisuitiprot, V.; Chaiwangrach, N.; Ingkaninan, K.; Waranuch, N. Cannabis Cannabinoid Res. 2022, 7, 537-547. Lewis Acid
  18. 18. CBD Content Degradation - Average CBD degradation of 20% 30 days at 37°C.3 - Effect of light alone resulted in 15% CBD degradation in 30 days.3 - Optimum CBD storage between pH 4-6.4 - Metal leaching – LA formation 3 Light Heat Bronsted Acid [3] Mazzetti, C.; Ferri, E.; Pozzi, M.; Labra, M. Sci. Rep. 2020, 10, 3697. [4] Jaidee, W.; Siridechakorn, I.; Nessopa, S.; Wisuitiprot, V.; Chaiwangrach, N.; Ingkaninan, K.; Waranuch, N. Cannabis Cannabinoid Res. 2022, 7, 537-547. Lewis Acid
  19. 19. CBD Degradation - Anecdotal evidence supports the degradation of CBD solution with multiple reports of CBD e-liquids and solutions turning purple. [5] Reddit. CBD E-juice turned purple in the tankhttps://www.reddit.com/r/Vaping/comments/e0z2ru/cbd_ejuice_turned_purple_in_the_tank/ (Accessed March, 2022). Sourced from Reddit5 4
  20. 20. The Beam Test - A classical colourimetric test for the identification of hashish using ethanolic KOH (5%).6 [6] Beam, W. Chemistry section in Fourth Report of Wellcome Tropical Research Laboratories, part B. Khartoum, 1911; pp 25. 5
  21. 21. The Beam Test - A classical colourimetric test for the identification of hashish using ethanolic KOH (5%).6 Cannabinoids responsible for a positive test result7,8 [6] Beam, W. Chemistry section in Fourth Report of Wellcome Tropical Research Laboratories, part B. Khartoum, 1911; pp 25. [7] Adams, R. Marijuana. Harvey Lecture. Bull. N. Y. Acad. Med. 1942, 18 (11), 705-730. [8] Mechoulam, R.; Ben-Zvi, Z.; Gaoni, Y. Hashish-XIII. On the nature of the beam test. Tetrahedron, 1968, 5615-5624. 5
  22. 22. Tracking HU-331 Concentration 𝒉𝒗 (65% PG, 30% VG, 5% CBD) Mass Chromatogram 6
  23. 23. R² = 0.9933 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 0 10 20 30 40 50 60 HU-331 Degradation Relative to T 0 Time (min) Light Dark Storage Conditions HU-331 Rate of Decomposition 45% reduction in [HU-331] after 60 minutes exposure to white light - Is HU-331 degradation a light-driven process? HU-331 (0.23 mg) + dinitrobenzene internal standard in DMSO-d6 (0.5 mL) 7
  24. 24. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 380 430 480 530 580 630 680 Absorbance (A) Wavelength (nm) CBD HU-331 and CBD at 0.5 mg mL-1 in IPA. Λmax of HU-331, 413 nm. 448 nm light emission Complete degradation of HU-331 in under 15 minutes Role of HU-331 vs CBD 8
  25. 25. 448 nm light emission HU-331 responsible for the colouration? 48 h Role of HU-331 vs CBD 9
  26. 26. Presence of HU-331 anion? 48 h 10 No HU-331 anion at high concentration (>10 mg HU-331)
  27. 27. Presence of HU-331 anion? 48 h 11 No HU-331 anion at high concentration (>10 mg HU-331) – what is purple?
  28. 28. Computational Exploration of Absorption 12 - TD-DFT-calculated visible light absorption spectra for HU-331 and its anion.
  29. 29. Computational Exploration of Absorption 12 - TD-DFT-calculated visible light absorption spectra for HU-331 and its anion. - Experimental data (linear) and calculated data (bar) closely correspond upon deprotonation of the hydroxyquinone.
  30. 30. 13 Computational Exploration of Absorption 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟒 Energy HU-331 𝝅𝑯𝑩𝒒𝟑
  31. 31. 14 Computational Exploration of Absorption 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟑 𝝅𝑯𝑩𝒒𝟒 H+    𝝅𝑻 𝝅𝑯𝑩𝒒𝟑 95% < 5% 𝝅𝑯𝑩𝒒𝟒 𝒏𝑶𝟐 Energy HU-331 HU-331
  32. 32. 15 Computational Exploration of Absorption 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟒 Energy HU-331 - How would absorption calculations change when the structure of HU-331 is altered? Olivetol-HQ 𝝅𝑯𝑩𝒒𝟑
  33. 33. 16 ∆𝐸~400𝑐𝑚−1 ∆𝐸~3000𝑐𝑚−1 HU-331 3 11 Olivetol-HQ Computational Exploration of Absorption 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟒 Energy HU-331 𝝅𝑯𝑩𝒒𝟑
  34. 34. 17 Computational Exploration of Absorption HU-331 anion 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟑 𝝅𝑯𝑩𝒒𝟒 H+    𝝅𝑻 𝝅𝑯𝑩𝒒𝟑 95% < 5% 𝝅𝑯𝑩𝒒𝟒 𝒏𝑶𝟐 Energy HU-331 HU-331 Olivetol-HQ anion
  35. 35. 17 Computational Exploration of Absorption HU-331 anion 𝝅𝑻 𝒏𝑶𝟒    40% 15% 35% 𝝅𝑯𝑩𝒒𝟑 𝝅𝑯𝑩𝒒𝟒 H+    𝝅𝑻 𝝅𝑯𝑩𝒒𝟑 95% < 5% 𝝅𝑯𝑩𝒒𝟒 𝒏𝑶𝟐 Energy HU-331 HU-331 Olivetol-HQ anion Structure of terpene is irrelevant
  36. 36. 18 Comparison to Experimental Data HU-331 anion Olivetol-HQ anion
  37. 37. 18 Comparison to Experimental Data HU-331 anion 0 0.1 0.2 0.3 0.4 0.5 0.6 380 480 580 680 Absorbance (A) Wavenlength (nm) HU-331 Anion Olivetol Hydroxyquinone Anion Olivetol-HQ anion 520 nm 540 nm
  38. 38. Conclusions - Likely that most olivetol-based structures which can from the hydroxyquinone will result in a purple colour. Cannabinoids responsible for a positive test result5 [6] Beam, W. Chemistry section in Fourth Report of Wellcome Tropical Research Laboratories, part B. Khartoum, 1911; pp 25. [7] Adams, R. Marijuana. Harvey Lecture. Bull. N. Y. Acad. Med. 1942, 18 (11), 705-730. [8] Mechoulam, R.; Ben-Zvi, Z.; Gaoni, Y. Hashish-XIII. On the nature of the beam test. Tetrahedron, 1968, 5615-5624. 19
  39. 39. Conclusions - Likely that most olivetol-based structures which can from the hydroxyquinone will result in a purple colour. Example quinones responsible for the purple colouration6 Cannabinoids responsible for a positive test result5 [6] Beam, W. Chemistry section in Fourth Report of Wellcome Tropical Research Laboratories, part B. Khartoum, 1911; pp 25. [7] Adams, R. Marijuana. Harvey Lecture. Bull. N. Y. Acad. Med. 1942, 18 (11), 705-730. [8] Mechoulam, R.; Ben-Zvi, Z.; Gaoni, Y. Hashish-XIII. On the nature of the beam test. Tetrahedron, 1968, 5615-5624. 19
  40. 40. Conclusions: Tracking HU-331 Concentration 𝒉𝒗 (65% PG, 30% VG, 5% CBD) Mass Chromatogram 20
  41. 41. University of British Columbia: Prof Glenn Sammis – Supervisor Prof Tao Huan Prof Pierre Kennepohl Dr Maria Ezhova Acknowledgements DELIC Labs: Kendra Payne Sajni Shah Dr Eric Janusson Dr Tom Dupree Amanda Assen Dingding Xuan Ali Wasti 20
  42. 42. Thank you! Questions?

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