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DuoSpray™ IonizationA Novel Approach to Analyzing the California Mandated List of Pesticides in Cannabis
Robert Di Lorenzo, PhDApplication Support Scientist
Diana TranApplication Support Scientist NACRW 2018
RUO-MKT-11-8253-A
© 2018 AB Sciex
• Market Overview
‒ State Regulations
• SCIEX Technology
• Analytical Data
‒ Meeting California requirements for testing
‒ Value Added Analysis
• Q & A
Overview
© 2018 AB Sciex
Introduction
www.marijuanapacking.com
www.marijuanastocks.com
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• State law determines which pesticides are to be tested and what the
appropriate action limits are in Cannabis
• Oregon has action limits for 59 pesticides and limits are the same for
flower and concentrates
• California’s new pesticide testing requirements contain 65 compounds
‒ 7 additional compounds compared to Oregon
‒ CA pesticide panel excludes MGK-264
‒ Category I (detect or non-detected)
‒ Category II (has specified action limits for different cannabis matrices)
Pesticide Testing Regulations
Introduction
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Introduction
OR Pesticide Residue Panel
https://public.health.oregon.gov
Abamectin Dimethoate Naled
Acephate Ethoprophos Oxamyl
Acequinocyl Etofenoprox Paclobutrazol
Acetamiprid Etoxazole Permethrins
Aldicarb Fenozycarb Phosmet
Azoxystrobin Fenpyroximate Piperonyl Butoxide
Bifenazate Fipronil Prallethrin
Bifenthrin Flonicamid Propiconazole
Boscalid Fludioxonil Propoxur
Carbaryl Hexythiazox Pyrethrins
Carbofuran Imazalil Pyridaben
Chlorantraniliprole Imidacloprid Spinosad
Chlorfenapyr Kresoxim-methyl Spiromesifen
Chlorpyrifos Malathion Spirotetramat
Clofentezine Metalaxyl Spiroxamine
Cyfluthrin Methiocarb Tebuconazole
Cypermethrin Methomyl Thiacloprid
Daminozide Methyl Parathion Thiamethoxam
Dichlorvos MGK-264 Trifloxystrobin
Diazinon Myclobutanil
https://swamis420.wordpress.com/2012/06/26/tropicana-strain-review/
Additional CA PesticidesCaptan Mevinphos
Chlordane Pentachloronitrobenzene
Coumaphos Spinetoram
Dimethomorph
Historically analyzed by GC-MS
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Introduction
Category I Pesticides
https://swamis420.wordpress.com/2012/06/26/tropicana-strain-review/
Category II Pesticides
Aldicarb Coumaphos Etofenoprox Mevinphos
Carbofuran Daminozide Fipronil Paclobutrazol
Chlordane Dichlorvos Imazalil Propoxur
Chlorfenapyr Dimethoate Methiocarb Spiroxamine
Chlorpyrifos Ethopro(phos) Methyl Parathion Thiacloprid
Abamectin Chlordantraniliprole Fludioxonil PCNB Spirotetramat
Acephate Clofentezine Hexythiazox Permethrin Tebuconazole
Acequinocyl Cyfluthrin Imidacloprid Phosmet Thiamethoxam
Acetamiprid Cypermethrin Kresoxim-methyl Piperonylbutoxide Trifloxystrobin
Azoxystrobin Diazinon Malathion Prallethrin
Bifenazate Dimethomorph Metalaxyl Propiconzaole
Bifenthrin Etoxazole Methomyl Pyrethrins
Boscalid Fenhexamid Myclobutanil Pyridaben
Captan Fenpyroximate Naled Spinosad
Carbaryl Flonicamid Oxamyl Spiromesifen
No CA defined limits
CA defined action limits
Analyzed via APCI
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Canadian List – Lots of Pesticides, Low Levels
Introduction
• 95 Pesticides (32 new) on Health Canada draft list for cannabis and related products
• Generally 20-40x lower
MRLs than Oregon‒ Many at 10 ppb in matrix
• Regulate for fresh
flower and oil in
addition to dried
product
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Introduction
Compound MRL (ppm)
Allethrin 0.1
Azadirachtin 0.5
Benzovindiflupyr† 0.01
Buprofezin 0.01
Clothianidin 0.025
Cyantranilipole 0.01
Cyprodinil* 0.01
Deltamethrin* 12.5
Dinotefuran 0.05
Dodemorph 0.05
Endosulfan sulfate 0.5
Endosulfan-alpha 0.1
Endosulfan-beta 0.5
Etridiazol 0.01
Fensulfothion 0.01
Fenthion 0.01
Compound MRL (ppm)
Fenvalerate* 3.75
Fluopyram 0.01
Iprodione 0.5
Kinoprene† 0.05
Methoprene 1
Novaluron 0.025
Phenothrin 0.025
Pirimicarb 0.01
Pyraclostrobin 0.01
Resmethrin 0.05
Spirodiclofen 0.01
Tebufenozide 0.01
Teflubenzuron 0.025
Tetrachlorvinphos 0.01
Tetramethrin 0.05
Thiophanate-methyl* 0.025
*- Minimum value in cases where MRL in flower is not yet determined
†- Few commercial sources for standard
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• Pesticide residue testing
is required by the state -
variable
• Lack of affordable in-
house LC-MS expertise
• Method development
takes too long
Challenges for a new cannabis testing lab
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Questions, we had to ask ourselves…
Motivation
Do you really need a GC and LC to run the entire panel?
Are all of those pesticides amenable to LC-MS analysis?
Can they all be done by ESI?
Problematic compounds on the California List of pesticides:
• Captan (low sensitivity)
• Chlordane (APCI Ionization)
• Pentachloronitrobenzene (PCNB) (APCI Ionization)
© 2018 AB Sciex
SCIEX Hardware
LC
FLOW
ESI
APCI
Corona Pin
ESI Spray
Flow diversion to either ionization
mode for APCI/ESI analysis
IonDrive™
QJet Guide
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Pesticides
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• SOP
‒ Sample Preparation
‒ System Performance Testing
• Acquisition Method
‒ Optimized Parameters
‒ Plug and play
‒ LC Separation of interferences
‒ Compatibility on two instruments
• Quantitation Method
‒ Internal STD designations
‒ Calibrators and quality controls
• Data Analysis
‒ Verified LOQ analysis for pesticides and cannabinoids
• Report Template
vMethod™ Application for Quantitation of Pesticide Residues and
Cannabinoids in Cannabis Matrices v1.0
Detailed SOP
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• 1 gram of homogenized flower is vortexed in
10 mL of Acetonitrile
• Sonicated for 15 minutes
• Centrifuged at 4000 rpm
• Extracts frozen, supernatant removed
• Injection volume of 1 of µL for LC-MS/MS
analysis
‒ The majority of the pesticides will have to
be detuned
‒ Although the additional sensitivity is
needed for Captan and PCNB to meet the
CA Action Limits.
Cannabis Flower Extraction
Pesticide MRM Analysis
Simple Solvent Extraction
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Pesticide Chromatography – Oregon List
Kinetex Biphenyl, 4.6x150mm (16 minute gradient)
Acephate
Avermectin B1aAvermectin B1b
Acequinocyl
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Using TCLP Chlordane and APCI Negative Ionization…
Chlordane
• Chlordane has 8 chlorine atoms
• Typical chlorinated pattern
• Chlordane underwent in source
fragmentation with APCI
M –HCl-
M -2HCl-
M -3HCl-
M -4HCl-
Cis/trans Chlordane are the
most abundant in mix
M
Q1 Scan
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Using RESTEK PCNB Standard with APCI Negative Ionization
PCNB
Q1 Scan
• PCNB has 5 chlorine atoms
• Typical chlorinated pattern
• PCNB also underwent in source
fragmentation with APCI
• Loss of HCl-
• Forms an ammonium adduct
Product Ion Scan
(MS2)
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Luna Omega Polar C18, 4.6x150mm (30 minute gradient)
Pesticide Chromatography – California List
APCI APCI
Analytical flow is diverted to APCI Analytical flow is diverted to APCI
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Analyzed in ESI
Parathion Methyl
Unspiked Flower
Extract
0.05 ppm in flower
1 ppb in vial
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
1 ppm in flower
20 ppb in vial
0.05 – 1 ppm in flower
1-20 ppb in vial
R2 = 0.9990
Solvent Standard
20 ppb in vial
CA- Schedule 1
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Analyzed in ESI
Cyfluthrin
Unspiked Flower
Extract
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
Solvent Standard
20 ppb in vial
1 ppm in flower
20 ppb in vial
0.05 ppm in flower
1 ppb in vial 0.05– 1 ppm in flower
1-20 ppb in vial
CA- 2 ppm
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Analyzed in ESI Ionization
Coumaphos
Unspiked Flower
Extract
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
Solvent Standard
20 ppb in vial
0.05 ppm in flower
1 ppb in vial
1 ppm in flower
20 ppb in vial
0.05 – 1 ppm in flower
1-20 ppb in vial
R2 = 0.9989
CA- Schedule 1
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Analyzed in APCI
Pentachloronitrobenzene
Unspiked Flower
Extract
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
Solvent Standard
20 ppb in vial
1 ppm in flower
20 ppb in vial
0.05 ppm in flower
1 ppb in vial 0.05 – 1 ppm in flower
1-20 ppb in vial
R2 = 0.9755 with first pointFirst STD can be
excluded as LOD
First STD can be
excludedCA- 100 ppb
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Analyzed in ESI
Captan
Unspiked flower
extract
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
1 ppm in flower
20 ppb in vial
5 ppm in flower
100 ppb in vial
Unspiked flower
extract
0.1 ppm in flower
2 ppb in vial
0.5 ppm in flower
10 ppb in vial
1 ppm in flower
20 ppb in vial
5 ppm in flower
100 ppb in vial
MRM 1
MRM 2
CA Action Limit is 0.7 ppm for
inhalables
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Analyzed in APCI
Chlordane
CA – Schedule 1
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Mycotoxins – Linearity and Reproducibility
Aflatoxin B1: r > 0.994, CV(n=4) = 5.76% at 12.5 ppt
Aflatoxin B2: r > 0.994, CV(n=4) = 8.71% at 12.5 ppt
Aflatoxin G1: r > 0.994, CV(n=4) = 8.12% at 12.5 ppt
Aflatoxin G2: r > 0.996, CV(n=4) = 7.59% at 12.5 ppt
Replicates at 1.9 ppb equivalent
Current Mycotoxins MRL = 20 ppb
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For when you need a little bit more…
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What is MS3?
QTRAP Analysis
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Hardware
QTRAP Analysis
2. Q2
fragments
precursor ion
with no low
mass cut-off
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MS3 for Quantitation
QTRAP Analysis
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What can MS3 do for Cannabis matrices? Using Pyrethrin Pyrethrin I as an example…
QTRAP Analysis
MRM of Pyrethrin Pyrethrin I
MS3 of Pyrethrin Pyrethrin I
Distinguishes real Pyrethrin Pyrethrin from isobaric interferences
Flower Blank
Flower Blank
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Cyfluthrin MS3 in Cannabis Flower Extract
QTRAP Analysis
Flower Blank
Flower Blank
2 ppb in vial
2 ppb in vial
10 ppb in vial
10 ppb in vial
20 ppb in vial
20 ppb in vial
MRM of Cyflutrhin
MS3 of Cyfluthrin
11 ppb
in matrix
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Changing Landscape – 100 ppb MRL for Category I
Category I Pesticides
https://swamis420.wordpress.com/2012/06/26/tropicana-strain-review/
Category II Pesticides
Aldicarb Coumaphos Etofenoprox Mevinphos
Carbofuran Daminozide Fipronil Paclobutrazol
Chlordane Dichlorvos Imazalil Propoxur
Chlorfenapyr Dimethoate Methiocarb Spiroxamine
Chlorpyrifos Ethopro(phos) Methyl Parathion Thiacloprid
Abamectin Chlordantraniliprole Fludioxonil PCNB Spirotetramat
Acephate Clofentezine Hexythiazox Permethrin Tebuconazole
Acequinocyl Cyfluthrin Imidacloprid Phosmet Thiamethoxam
Acetamiprid Cypermethrin Kresoxim-methyl Piperonylbutoxide Trifloxystrobin
Azoxystrobin Diazinon Malathion Prallethrin
Bifenazate Dimethomorph Metalaxyl Propiconzaole
Bifenthrin Etoxazole Methomyl Pyrethrins
Boscalid Fenhexamid Myclobutanil Pyridaben
Captan Fenpyroximate Naled Spinosad
Carbaryl Flonicamid Oxamyl Spiromesifen
All at 100 ppb MRL
CA defined action limits
Analyzed through APCI
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IonDrive™ Technology Key Innovations
IonDrive™ Turbo V Source IonDrive™ QJet Guide IonDrive™ HE Detector
Driving Performance by Driving More Ions While Maintaining Ruggedness
Make More Ions Collect More Ions Detect More Ions
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• Larger diameter (11 mm) heaters
• Optimized geometry
• More efficient heat transfer
• Covers a larger cross-section of the
spray cone
• Wider “sweet spot” when optimizing
probe position
• More robust against fluctuations in
gas flow dynamics, and source to
source differences
IonDrive™ Turbo V Source – Make More Ions
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IonDrive™ Turbo V Source – Make More Ions
Original Turbo V™ IonDrive™ Turbo V
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IonDrive™ Turbo V Source – Make More Ions
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Cannabis Matrix
Mycotoxins
TerpenesCannabinoids
PesticidesTotal
Cannabis
Solution
How many instruments/extracts/analyses are necessary?
Extract + Cleanup 1 & 2
LC-MS/MS
GC-MS(/MS)Extract + Cleanup 3
LC-MS/MS
Extract + Cleanup 4
LC-UV
2-injectionsExtract + Cleanup 5
GC-FID (or MS)
1 Extract
1 Column
1 Solvent System
1 Instrument
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
Potency
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• Current regulations require the quantitative
reporting of cannabinoid compounds
‒ Concentrations differ greatly between strains
and analytes
‒ High abundance
‒ THC, THCA, CBD, CBDA
‒ Low abundance
‒ CBG, CBN, CBC, THCV, CBDV…
‒ Concentrations > 90% by weight to < 0.01%,
depending on analyte/strain
• Commonly performed by LC-UV
‒ Two injections needed
‒ One for high abundance compounds
‒ One for low abundance compounds
‒ Concentration range: 0.05 – 30% w/w
Potency Analysis
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• Dilute extract 1:6
‒ Use for Pesticides, Aflatoxins
• Dilute serially further 2000x
‒ Use for Potency
Sample Dilution
Current vMethod Sample Prep Protocol
Calibration standards
Concentration in Extract Equivalent Concentration in Sample
10 ppb 0.3%
50 ppb 1.5%
200 ppb 6%
500 ppb 15%
800 ppb 24%
1000 ppb 30%
Need to go lower (< 0.05%)
Need to go higher (~100%)
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
• Need to increase linear dynamic range (LDR) to
the low end and to the high end
• Instead of diluting 1:6 extract 2000x, only dilute
200x
‒ Large dilution will still eliminate all matrix effects
• 10 ppb standard now becomes equivalent to
0.03% in sample
‒ Low end reached
• Extend standard concentrations to ~33 ppm
which would be equivalent to 99% in sample
‒ High end reached
• Problem: ESI is prone to saturation at high end
Sample Dilution
Modifications to extend potency range (0.03%-100%)
New Calibration Standards
Concentration in Extract Equivalent Concentration in Sample
10 ppb 0.03%
50 ppb 0.15%
200 ppb 0.6%
500 ppb 1.5%
800 ppb 2.4%
1200 ppb 3.0%
3000 ppb 9.0%
6000 ppb 18%
10 000 ppb 30%
20 000 ppb 60%
30 000 ppb 90%
Solution: Use APCI instead of ESI
Bonus: May be able to simultaneously
analyze other compound classes
(Hint: It’s terpenes)
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CASE 2
CASE 3
MRM Monitoring
Modifications to extend potency range (0.03%-100%)
• Many possible MRM transitions can be monitored for each analyte
‒ Varying degree of sensitivity seen for each transition
‒ Currently only 2 transitions are used per analyte
‒ 1 for quantitation, 1 for confirmation
• May saturate detector at high end of calibration range
THCA
9 Transitions
THC
7 Transitions
CBD
6 Transitions
CBDA
8 Transitions
Analyze entire
calibration range with
one MRM per analyte
Full LDR?
Stop
Yes
Use one MRM for high
end and another for
low end of calibration No
Full LDR?
Stop
Yes
Detune DP and CE to
eliminate saturation at
high end of calibration
Full LDR?
Stop
Yes
No
Solution Flowchart
CBDV
CBD
CBDACBGA
CBG
THCV
CBN
CBC
THC
THCA
CASE 1
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THC – Case 1 - Full range in one transition
Results – Extended Linear Dynamic Range
Linear Range
10-30 000 ppb in vial
0.03-90% in sample
R2 = 0.999
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THCA – Case 2 – Two transitions required
Results – Extended Linear Dynamic Range
Linear Range
10-10 000 ppb in vial
0.03-30% in sample
R2 = 0.995
Linear Range
1200-30 000 ppb in vial
3.6-90% in sample
R2 = 0.995
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CBDV – Case 3 – Detuning required
Results – Extended Linear Dynamic Range
Linear Range
10-10 000 ppb in vial
0.03-30% in sample
R2 = 0.999
• All transitions monitored for CBDV saturate at the high end of the curve
• Need to further detune DP and CE to extend range to 100%
• Sample concentration is expected to be below 1%
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
• Calibration extended to effective concentrations
of 0.03%-90% by weight in sample
‒ All but CBDV and THCV, which are linear
between 0.03-30%
‒ These are not expected to be above 1%
• APCI less prone to saturation than ESI
‒ Some analytes only need one transition monitored
• Excellent reproducibility and accuracy
‒ All calibrations R2 ≥ 0.995
‒ CV < 10%
‒ Most < 5%
‒ Accuracy
‒ Within 20% for 0.03% calibration standard
‒ Within 10% for all other standards
‒ Most within 5%
CannabinoidCalibration
Range 1R2 Calibration
Range 2R2
THC 0.03-90% 0.999
THCA 0.03-30% 0.995 3.6-90% 0.995
CBD 0.03-90% 0.999
CBDA 0.03-3.6% 0.999 3.6-90% 0.998
CBG 0.03-90% 0.999
CBGA 0.03-9% 0.999 9-90% 0.998
CBN 0.03-90% 0.999
CBC 0.03-30% 0.998 0.15-90% 0.999
CBDV 0.03-30% 0.999
THCV 0.03-30% 0.999
Summary
• Potency analysis can be performed in
a single injection
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Terpenes
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• 200+ terpenes have been identified in
Cannabis
‒ Unique strains presenting varying terpene
profiles
‒ Contribute primarily to flavor and aroma
‒ Potentially have beneficial health outcomes
‒ ‘Entourage’ effect
• Only Nevada requires terpene analysis
‒ Consumers and aficionados are highly
interested in terpene profiles
‒ Growers interested in profiling terpenes as a
metric for product quality and batch
reproducibility
Terpene Profiling
© 2018 AB Sciex
• Six commonly reported terpenes
• Not amenable to ESI
‒ Usually analyzed by GC
• Quite responsive to APCI
• Challenging separation
‒ Series of structural isomers with identical MRM
transitions
‒ All relevant compounds must be separated
chromatographically
• Previously shown to be possible by LC-
MS/MS, but using a different column and
solvent system than used in vMethod
Terpene Profiling
Linalool β-Myrcene α-Pinene Limonene α-Humuleneβ-Caryophyllene
© 2018 AB Sciex
Chromatography in solvent
Linalool
b-Myrcene
a-Pinene
Limonene a-Humulene
b-Caryophyllene
CBDCBDA
CBGA
CBG
CBDV
THCV
CBC
THCA
THC
© 2018 AB Sciex
• Extending the terpene profiling from 6 to 20+
terpenes
• Still using same column, solvent system and
extract as existing vMethod™
• Mix of monoterpenes, sesquiterpenes, their
hydroxylated analogues and some miscellaneous
aroma compounds
• Challenging chromatography achieved with
baseline separation of nearly all isomers
‒ Most abundant and most commonly observed
terpenes included in method
Extended ProfilingLinalool
Geraniol
Eucalyptol
Isop
ule
gol
Terp
ineol
R,S
-Born
eo
l
R,S-Pulegone
Ocim
ene
α,β-Pinene
Limonene (137/95)
Eudesmol
α-Bisabolol
Guaiol (205/149)
Myrc
ene
β-Caryophyllene
β-Humulene
Menth
ol (1
39
/97)
Citro
nello
l(1
57/8
3)
R,S-Pulegone (153/93)
Caryophyllene oxide (221/203)
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
• Leveraging the power of APCI to
simultaneously analyze for
compounds natively produced in
Cannabis‒ Namely terpenes and cannabinoids
‒ Phenotype strains using multivariate
regression tools
• Potential to qualitatively and
quantitatively assess similarities and
differences between strains
‒ Consumer standpoint
‒ Health benefit standpoint
Cannabis Phenotyping
SCIEX Users Meeting – KC Hyland
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
• ESI and APCI used effectively on diluted
extracts to meet MRLs for California
mandated list of pesticides
• Mycotoxins seamlessly added to verified
analysis method
Pesticides and Mycotoxins
• Exploiting the power of APCI to ionize non-
polar compounds with minimal matrix effects
and large LDR
• Linear dynamic range extended for potency
analysis
‒ Simultaneous analysis of low and high level
cannabinoids in one injection
‒ Concentration range between 0.03-90% w/w
‒ Potential to extend LDR further
• Quantitative analysis of most abundant
terpenes
• Challenging separation achieved for extended
list of terpene isomers
Cannabinoids and Terpenes
Summary
All analyses performed using a
single extract
All analyses able to be performed on
a single LC-MS/MS system
© 2017 AB SciexCOMPANY CONFIDENTIAL & PROPRIETARY
Questions?
© 2018 AB Sciex
For Research Use Only. Not for use in diagnostic procedures.
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AB SCIEX™ is being used under license.