Entheogens, frequently used interchangeably with hallucinogens and psychedelics, are naturally occurring psychoactive substances. Historically, these substances have been used to induce a change of perception, mood, consciousness, or behavior for the purposes of spiritual development or social enrichment. In addition to these uses, these substances have become strong candidates for alternative medicines for the treatment of psychological disorders. Cannabis, a more common entheogen, has paved the way for decriminalization and legalization of other substances, such as psilocin, psilocybin, MDMA, and LSD to be used for clinical treatments. While these compounds in many places are still classified as Schedule I substances under the Controlled Substances Act, other entheogenic plants with different psychoactive compounds are not, such as Mitragyna speciosa and Amanita muscaria, which already appear on the market in the form of edibles, extracts, or powders. With the lack of standardized methods for these psychoactive targets, and with the rise of legalization and clinical treatment centers, it is important to understand this emerging market from a safety and quality perspective. This session will focus on emerging products, testing targets, sample preparation, method development, and analytical challenges. Attendees of this session will learn technical steps that need to be considered to develop robust, reproducible analytical methods, and a quality assurance program to expand into this new and evolving area of testing.

1. Have a Good Trip?
How to Analyze
Psychedelics
Dr. Markus Roggen
PittCon, Feb. 27th 2024

2. Callum Teevens
3rd year student at the University of Victoria
Pursuing a bachelors degree in Bio-Psyc neurology
Introduction
Dr. Markus Roggen
Founder of Controlled Chemistry

3. Controlled Chemistry
• A new innovator in cannabis and psychedelic chemistry, building on
CBDV and Delic Labs' legacies
• Located within the chemistry department of University of British
Columbia, Canada
• Combining government grants and industry collaborations to support
independent, impactful research
• Utilizing CBDV and Delic Labs' successes to inspire and revolutionize
future industrial applications in cannabis and psychedelic chemistry

4. Table of contents
• Psychedelic classes
• Testing
• Challenges
• Summary
• References

5. Psychedelics
The Compounds of Mystery

6. Overview of Psychedelics
• Serotonergic hallucinogens
• 5-HT2A receptor agonists with
a higher affinity than
serotonin
• Mechanism is not fully
understood

7. Overview of Psychedelics
• Serotonergic hallucinogens
• 5-HT2A receptor agonists with
a higher affinity than
serotonin
• Mechanism is not fully
understood
• Many different forms
• Grouped into three families
N
H
OH
N
N
H
O
H
N
P
O
HO
O
N
H
N
N
H
N
O
N
H
OH
N
N
H
O
N
H
N NH2 NH2
O
O
O
Br
O
O
O
O
N N
NH
O
H
Psilocybin Psilocin DMT
4-OH-MET 5-OH-DMT Ibogaine
MDMA 2C-B Mescaline
LSD

8. Tryptamines
• Core moiety of indolamine
• Structurally related to L-tryptophan
• Naturally occurring alkaloids and
synthetically made
• Neurotransmitters, hormones
and 5-HT2A receptor agonists
• Melatonin, Serotonin, Psilocin,
DMT, 5-MeO DMT and Ibogaine
N
H
OH
N
Psilocin
N
H
NH2
Serotonin
HO
N
H
HN
Melatonin
O
O
N
H
L-Tryptophan
OH
NH2
O

9. Phenethylamines
• Amine connected to a benzene ring via an
ethyl bridge
• Phenylamine backbone
• Naturally occurring and synthetically made
• Dopamine, (nor)epinephrine, 2C-B, mescaline
and 4-OH-MET, MDMA*
• Neurotransmitters, hormones and 5-
HT2A receptor agonists
NH2
Phenethylamine
NH2
HO
OH Dopamine
NH2
O
O
O Mescaline
H
N
O
O
MDMA

10. Lysergamides
• Lysergic acid backbone
• Contains tryptamine and
phenylethylamine groups with
a carboxamide
• Have a greater affinity for 5-HT2A
receptors
• LSD and derivatives
• Ergotamine and Methysergide
HO N
NH
O
H
Lysergic Acid
NH
H
N
O
NH
O
N
O
N
HOH
O
Ergotamine
H
N
N
O
NH
HO
Methysergide
HO N
NH
O
H
Lysergic Acid
N N
NH
O
H
LSD

11. Psychedelic Testing
The How and Why

12. Licensing in Canada
• Regulated under the controlled
drugs and substances act (S.C.
1996, c.19)
• Most psychedelics are class III
substances
• A license is required by Health
Canada
• Prohibited unless granted
exemption under section 56 of
CDSA
Type Duration Actual Duration
New applications 270 days ~ 660 days
Renewals 90 days ~470 days
Amendments 45 days ~200 days
Change in
Personnel
45 days ~470 days

13. Regulations Outside of Canada
US:
• Schedule 1
• Enforced by the DEA
• Research requires approval by FDA
and then by institutional review
board

14. Regulations Outside of Canada
US:
• Schedule 1
• Enforced by the DEA
• Research requires approval by FDA
and then by institutional review
board
Europe:
• Legal status varies by country
• Require approval from
European Medicines Agency

15. Types of Testing
• Public Safety Testing
• Commercial product testing
• Police/forensic testing
• Scientific research

16. The work horse!
High Performance Liquid Chromatography (HPLC)
• Optical Detectors
• Mass spec
• DAD/PAD
• UHPLC
• MS/MS

17. Other analytical Instruments
• Gas Chromatography (GC)
• Nuclear Magnetic
Spec (NMR)
• Infrared Spec (IR)
• Capillary zone
electrophoresis (CZE)

18. Tryptamine Testing

19. Sample Prep: Extraction
• Dried, Pulverized or lyophilized before extraction
Solvent Co-solvent/Acid Mechanical Temp Time
Acetic Acid
I. Suspension
II. Sonication
III.Shaking
IV.Vortex
<70 ˚C
1 - 48
hours
Water
Ethanol
Methanol
Methanol Acetic Acid
Methanol Water

20. Tryptamine Analysis
Most common (U)HPLC:
• Tandem Mass Spec
• UV, IR Fl, DAD
• Variety of matrices
GC is less used do to:
• Heating
• Required derivatization
• Lipid content
References: 19

21. Product
Samples
• System: HPLC-DAD or HPLC-MS/MS
• Column: C18 or Biphenyl
• Mobile Phase: Water/ACN with formic acid
System Column Mobile Phase
HPLC-ESI-MS RP-18 GP 50mM ammonium acetate and ACN mix (73:27)
HPLC-MS/MS C18 (A) Formic acid, ACN and water
UHPLC-MS/MS XR-ODS II column (A) 0.1% formic acid in water
(B) ACN
HPLC-PDA Biphenyl 90% (0.1% TFA in H20) and 10% (0.1% TFA in 2:1 ACN to methanol)
UHPLC-PDA/QDA HSS C18 (A) 95% (5mM ammonium formate)
5% (0.2% formic acid in ACN)
HPLC-DAD (220nm) Symmetry C18 10mM ammonium formate buffer (3.5ph): ACN (95:5 ratio %)
UHPLC-DAD Gemini C18 (A) 0.2% formic acid in water
(B) 0.2% formic acid in ACN
HILIC HPLC-DAD ZIC-pHILIC ACN-40mmol formate buffer ph 3.5
GC-MS capillary column
(95% dimethyl-5% diphenyl polysiloxane)
Helium gas

22. Biological
Samples
• System: UHPLC-MS/MS
• Column: More variation
• Mobile Phase: ACN/water + acid
Sample System Column Mobile Phase
Urine/Oral fluid
UHPLC-MS/MS
Water Oasis HLB Ammonium Formate and ACN
Blood
C18
(A) Water with 0.1% acetic acid
(B) Methanol with 0.1% acetic acid
(A) Water + 50 mM AcONH4
(B) Acetonitrile
Hair
HSS C18
HSS T3
Allure PFPP
(A) 0.1% formic acid + water
(B) ACN
(A) 0.3% formic acid + ACN
(B) 5mM ammonium formate

23. Phenethylamine Testing

24. Phenethylamine Analysis
• Broad range of chemicals with
different properties
• HPLC-DAD: used for
simple sample analysis
• UHPLC-MS/MS: used for
biological analysis

25. Product
Samples
• System: HPLC-DAD
• Column: Variety, C18
• Mobile Phase: ACN + phosphate buffer/acid
System Column Mobile Phase
HPLC-DAD ODS C18 MeOH + water (90:10 v/v)
HPLC-DAD Chromspher B with 1005 deactivated c18 0.05 phosphate buffer with ACN pH 3.2 (9:1)
UHPLC-MS/MS SB-C8 column (A) 0.1% formic acid in water
(B) 0.1% formic acid in MeOH
HPLC-DAD PKB100 20mM phosphate buffer and ACN
HPLC-DAD RP-18e (A) 20mM Phosphate buffer with triethylamine and
phosphoric acid
(B) ACN
HPLC-Fl 10 cm CN ACN + Water + triethylamine
HPLC-DAD PFP 100A (A) ACN
(B) Phosphoric acid + hexylamine

26. Biological
Samples
System: UHPLC-MS/MS
Column: C18
Mobile phase: ACN/water/methanol + acid
Matrix System Column Mobile phase
Hair
UHPLC-MS/MS
I. Phenylhexyl
II. Biphenal
III. BEH C18
A) 2 mM ammonium formate with 0.1% formic acid
B) ACN/methanol (50:50, v/v) and 1% eluent A.
A) 0.1% formic acid in water and acetonitrile
Urine/Saliva dC18 Ammonium formate buffer/acetonitrile (1:1)
Plasma HSS T3 C18 A) ammonium acetate (10 mM) supplemented with 0.1%
acetic acid
B) methanol containing 0.1% acetic acid
Blood/Urine
& Hair C18
Formate buffer 2 mM pH 3.0 and 20% acetonitrile

27. Lysergamide Testing

28. Lysergamide Analysis
• Does not require rigorous
extraction
• Heat and light sensitive
• GC and (U)HPLC, viable options
• HPLC: more researched needed

29. Product
Samples
System: HPLC-MS/MS
Column: Variety
Mobile phase: (A)0.1% formic acid in water
(B)0.1% formic acid in ACN or MeOH
System Column Mobile Phase
UHPLC-ESI-MS DB5-MS (A) water + formic acid + ammonium formate (2mmol/L)
(B) methanol + formic acid + ammonium formate (2mmol/L)
HPLC-ESI-QTOF-MS Biphenyl column (A) ACN + formic acid + 2mM ammonium formate in water
(B) formic acid + 2 mM ammonium formate in ACN
HPLC-ES-MS PFPP column (A) 0.1% formic acid in water
(B) 0.1% formic acid in acetonitrile
HPLC-MS/MS SB-C18 column acetonitrile + 0.05% formic acid
HPLC-ESI-MS and APPI-MS C18 column (A) 0.1% formic acid in water
(B) 1% formic acid in methanol
UHPLC-MS/MS SB-C18 column (A) 0.1% formic acid in water
(B) 0.1% formic acid in MeOH
UPLC with PDA and MS HSS T3 (A) 0.1% formic acid in water
(B) 0.1% formic acid in ACN
GC-MS Capillary DB-1HT Helium gas

30. Human
Samples
Type System Column Mobile phase
Hair
UHPLC-ESI-MS
UHPLC-MS/MS
Allure PFPP with C18 extend
Zorbax C18 RRHD
(A) 2mM ammonium formate with 0.2% formic acid in water
(B) 2mM ammonium formate with 0.2% formic acid in ACN
(A) 20 mM ammonium acetate with 0.1% formic acid in water)
(B) ACN
Urine/Saliva Biphenyl, Phenylhexyl,
RP 8S
water (40%) and acetonitrile (60%) containing 0.1% HCOOH and 2 mM of HCOONH4
(A) consisted of deionized water with 1% ACN, 0.1% formic acid, and 2 mM
ammonium formate
(B) 0.1% formic acid and 2 mM ammonium formate in CAN
(A) water with 0.1% formic acid and 2mmol/L ammonium formate
(B) methanol with 0.1% formic acid and 2mmol/L ammonium formate
(A) 2mM aq ammonium formate with 0.1% formic acid and 1%ACN
(B) 2mM ammonium formate in ACN/H20 mix with 0.1% formic acid and 1% water
Pursuit C18 &
C18XL turboflow
(A) ACN with 0.1% formic acid
(B) 2mM ammonium acetate buffer with 0.1% formic acid)
Plasma
(A) Eluent:5mM ammonium acetate in water with 0.10% formic acid
(B) Eluent: 5mM ammonium acetate in methanol with 0.50% formic acid
*10mM ammonium carbonate in water was used as buffer
System: UHPLC-ESI/MS or MS/MS
Column: C18 or Biphenyl
Mobile Phase: ACN/water + formic acid
and ammonium formate

31. Challenges
• Lack of standard method
• Sample preparation
• Analyte stability
• Detection limits
• Cost
• Licensing

32. Summary
HPLC-MS/MS:
• Preferred system for product
matrices
• Works with most psychedelics
• ACN/water + formic acid
• C18 or Biphenyl column

33. Summary
UHPLC-MS/MS:
• Preferred system for biological
samples
• Works with most psychedelics
• ACN/water + formic acid +
ammonium formate
• C18 or Biphenyl column
HPLC-MS/MS:
• Preferred system for product
matrices
• Works with most psychedelics
• ACN/water + formic acid
• C18 or Biphenyl column

34. The Next Steps
• Develop a standard method for
analysis
• Create a rapid extraction
method
• Create a cost-effective test
• License more labs

35. References
1) DOI: 10.1556/2054.2019.008
2) The Medical History and Use of Psychedelic Drugs: Ancient, Recent, and Present Events by Renshaw, William Brock, Southern Illinois University 2022
3) DOI: 10.1124/pr.115.011478
4) DOI: 10.1007/978-3-642-55214-4_96-1
5) DOI: 10.1016/j.neuropharm.2017.12.040
6) DOI: 10.1080/02791072.2019.1606472
7) DOI: 10.1038/npp.2017.84
8) DOI: 10.1073/pnas.1518377113
9) DOI: 10.1007/7854_2017_475
10) DOI: 10.1007/7854_2017_478
11) DOI: 10.3390/ijms21239279
12) DOI: 10.1007/s00204-015-1513-x
13) DOI: 10.3390/molecules28020855
14) UNODC New Psychoactive Substances Portal and International Collaborative Exercise Portal
15) DOI: 10.1016/S0893-133X(98)00135-3
16) DOI: 10.1038/npp.2017.86
17) DOI: 10.1016/j.jpba.2014.08.006
18) DOI: 10.1016/j.jchromb.2022.123202

36. Questions?
Dr. Markus Roggen
markus@complexbiotech.com

37. Thank You