Psychedelics are a diverse group of drugs that are known for their ability to alter consciousness, perception, mood, and thought. Detecting the presence, quantity and quality of these compounds is crucial to research development and involves various analytical tools such as High Performance Liquid Chromatography (HPLC) with optical or mass detectors, and other instruments types. These analytical tests are performed for a variety of reasons, including product, drug, or safety testing, all of which are subject to regulations and guidelines set by the licensing authorities. Besides the regulations, we face several other challenges with psychedelic analysis, such as the lack of standardized testing methods, difficulties in sample preparation, and analyte stability.
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How Long Is Your Trip? Analysing the Micros and Heroics of Psychedelics
1. Callum Teevens & Dr. Markus Roggen
May 30th, 2023
How Long Is Your Trip?
Analysing the Micros and Heroics of
Psychedelics
2. Callum Teevens
3rd year student at the University of Victoria
Pursuing a bachelors degree in Bio-Psyc neurology
Introduction
Dr. Markus Roggen
President / CSO of Delic Labs
3. Delic Labs
• 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
4. Table of contents
• Introduction
• History of psychedelics
• Psychedelic classes
• Testing
• Challenges
• Summary
• References
6. Ancient Tools
• First evidence dates to 5000
BCE
• Medicinal, ceremonial and
recreational uses
• Psilocybin, mescaline and
lysergic acid were commonly
used
• Combinations of plants
• Parallel... in different location
References: 1, 2
7. Promising Medicine
• Albert Hoffman: realizes
therapeutic properties
• Focused research: 1950s to
1960s
• Controlled Substances Act:
stopped work in 1970s
• War on Drugs: stigmatized
further
• Research Renaissance: in 2000s
References: 3, 4, 5
8. A Hopeful Future
Psychedelics show potential for:
• Mental health benefits
• Help for treatment resistant
patients
• Addiction treatment
Your Brain on Drugs!
References: 6, 7, 8
9. A Hopeful Future
Psychedelics have been found to:
• Increased neural connection
• Decrease default mode network
• Provide insight into
the human experience
• Physiologically safe and low risk
Your Brain on Drugs!
References: 6, 7, 8
10. Overview of Psychedelics
• Serotonergic hallucinogens
• Many different forms
• 5-HT2A receptor agonists with a
higher affinity than serotonin
• Mechanism is not fully
understood
• Grouped into three families
References: 9, 10, 11
11. Overview of Psychedelics
• Serotonergic hallucinogens
• Many different forms
• 5-HT2A receptor agonists with a
higher affinity than serotonin
• Mechanism is not fully
understood
• Grouped into three families
References: 9, 10, 11
12. Overview of Psychedelics
• Serotonergic hallucinogens
• Many different forms
• 5-HT2A receptor agonists with
a higher affinity than
serotonin
• Mechanism is not fully
understood
• 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
References: 9, 10, 11
13. 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
References: 12, 13
14. 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
References: 14, 15, 16
15. 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
References: 17, 18
17. 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 630 and
counting
Renewals 90 days ~470 days
Amendments 45 days ~200 days
Change in
Personnel
45 days ~470 days
18. Regulations Outside of Canada
US:
• Schedule 1
• Enforced by the DEA
• Research requires approval by FDA
and then by institutional review
board
19. 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
20. Types of Testing
• Public Safety Testing
• Commercial product testing
• Police/forensic testing
• Scientific research
21. The work horse!
High Performance Liquid Chromatography (HPLC)
• Optical Detectors
• Mass spec
• DAD/PAD
• UHPLC
• MS/MS
22. Other analytical Instruments
• Gas Chromatography (GC)
• Nuclear Magnetic
Spec (NMR)
• Infrared Spec (IR)
• Capillary zone
electrophoresis (CZE)
24. 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
25. 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
26. 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
27. System Column Mobile Phase
HPLC-ESI-MS RP-18 GP 50mM ammonium acetate and ACN mix
(73:27)
HPLC-MS/MS C18 Formic acid, ACN and water
UHPLC-MS/MS XR-ODS II column (A) 0.1% formic acid in water
(B) ACN
HPLC-PDA Biphenyl (A) 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 Helium gas
28. 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
30. Phenethylamine Analysis
• Broad range of chemicals with
different properties
• HPLC-DAD: used for
simple sample analysis
• UHPLC-MS/MS: used for
biological analysis
References: 20
31. 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
32. 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 phosphtae 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
33. 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
35. Lysergamide Analysis
• Does not require rigorous
extraction
• Heat and light sensitive
• GC and (U)HPLC, viable options
• HPLC: more researched needed
36. 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
37. 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-C8 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
38. 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
Plasma Pursuit C18 &
C18XL turboflow
(A) ACN with 0.1% formic acid
(B) 2mM ammonium acetate buffer with 0.1% formic acid)
(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
39. Challenges
• Lack of standard method
• Sample preparation
• Analyte stability
• Detection limits
• Cost
• Licensing
41. Summary
UHPLC-MS/MS:
• Preferred system for biological
samples
• Works with most psychedelics
• ACN/water + formic acid +
ammonium formate
• C18 or Biphenyl column
Portugal: Decriminalized all drugs in 2001, including psychedelics. Possession and use of small amounts of drugs are treated as a public health issue not a criminal offense. However, production, sale, and trafficking are still illegal.
Netherlands: Psychedelic mushrooms and truffles containing psilocybin are legal to purchase and consume in licensed "smart shops". However, the production and sale of these substances remain illegal, and possession outside of licensed smart shops is also illegal.
Spain: Spain has a "grey area" regarding the legality of psychedelic substances. Personal use and possession aren't a crime, but cultivation and distribution is illegal.
Germany: The use of psychedelics is illegal in Germany, with the exception of some limited medical and scientific research. Some German cities have decriminalized possession
United Kingdom: Psychedelics are classified as Class A drugs, the most serious drug classification. Possession, distribution, and production of these substances is illegal.
Quantifying and analyzing psychedelics poses several challenges due to various factors. One of the most significant hurdles is the lack of standardized analytical methods. As different psychedelics have distinct chemical structures, each requires a specific analytical method for precise quantification. For instance, certain methods may be more suitable for LSD analysis, while others may be more effective for psilocybin analysis. This lack of standardization can make it challenging for researchers to compare results across different studies.
Another obstacle in psychedelic analysis is determining the detection limits of the analytical method used. The detection limit refers to the lowest amount of a substance that can be reliably detected and measured using a particular method. For psychedelics, determining the detection limit can be particularly difficult due to the small doses used in clinical studies and their rapid metabolism and elimination from the body.
Sample preparation is also a crucial factor in accurately quantifying psychedelics. To ensure the purity and stability of the compound being analyzed, samples must be prepared carefully. This may involve several steps, including extraction, purification, and derivatization, and may require specialized equipment and time.
Matrix effects can also affect the accuracy of psychedelic quantification. Matrix effects occur when the presence of other compounds in the sample interferes with the analysis of the compound of interest. This can be especially challenging with biological samples like blood or urine, where the presence of other compounds can make it difficult to accurately quantify psychedelics.
Calibration standards are critical for precise quantification of psychedelics. These standards are used to create a calibration curve, which is used to determine the amount of a compound present in a sample. However, obtaining accurate and reliable calibration standards can be challenging due to the limited availability of pure compounds and the difficulties in synthesizing these compounds, particularly with psychedelics.
Licensing
