Ric Cuchetto
Objectives
Appreciate some of the obstacles states face when developing pesticide protocols for cannabis
Political
Scientific
Market-based
Basic understanding of technologies available
Physical limits
Sensitivity
Human limits
Process Feedback and Control
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OMMP July 1, 2016
66,880 patients
29,833 caregivers
Oregon Medical Marijuana Program
November 1998
Not for Profit patient providers:
Caregivers
Growers
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Implementation of recreational sales in OR and WA have spawned regulations that overlap or eclipse existing medical policy.
OMMP OLCC, OHA
ORELAP
Classes of pesticides Carbamate
EPA 531 HPLC derivitized
Organochlorine EPA 8081
GC-ECD
Pyrethroid EPA 8270
GC-MS
Organophosphate EPA 8140
GC-FPD
EPA Method language GCMS for semi-volatile compounds 1980’s technology
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GC-FID (for compliance?)
2/15/2018
Inaccuracies multiply at lower concentrations and with RT shift
“hydrocarbon burner”;non-specific detector
Thin Layer Chromatography
(TLC)Immunoassays
(ELIZA)Total Organic Halides
(TOX)
© 2017 The Cannabis Chemist ™ 5
This slide from Jack Cochran’s excellent blog at Restek
Lower cost technologies
GC: Co-extractives of cannabis
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Thanks to Rodger Voelker, PhD
MS vs MS/MSsingle quad (GC or LC) cannot detect low levels in complex matrices
triple quad (GC or LC) can
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Matrix effect becomes more pronounced as foodstuff gets more complex (less water, more oily)
Cannabis and hops behave more like spinach or citrus
Accurate ID and quantitation via MS/MS
Tandem Quad aka Triple Quad –The Typical Approach for QqQ
© 2017 The Cannabis Chemist ™ 8
Chromatogram of 195 Pesticides in Organic Honey
2/15/2018
Technology shift
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2013 – GC MSD $36k (refurbished) 2016 – HPLC MSTQ $300k+
new language/disciplineGC LC
new technologyMSD TQ
New cost of entry
Pesticides – new targetsAbamectin
(Avermectins B1a and B1b Insecticide/acaricide LC-FLD1; LC-MS/MS2
Bifenazate Acaricide LC1; LC-MS/MS2
Bifenthrin
(synthetic pyrethroid) Insecticide GC-ECD1; GC-MS/MS2
Cyfluthrin
(synthetic pyrethroid) Insecticide LC2 (WHO 2004); GC-MS/MS2
Daminozide(Alar) Plant growth regulator (PGR); fungicide UV Spectroscopy1; LC-MS/MS2
Etoxazole Acaricide GC-MS(/MS)1
Fenoxycarb Insecticide LC/UV1; LC-MS/MS2
Imazalil Fungicide GC-ECD1; LC-MS/MS2
Imidacloprid Insecticide LC-MS/MS2
Myclobutanil Fungicide GC-ECD; GC-NPD1; GC-MS/MS)2; LC-MS/MS2
Paclobutrazol Plant growth regulator (PGR); fungicide LC-MS/MS2
Pyrethrins Insecticide GC-ECD1
Spinosad Insecticide LC-MS/MS; immunoassay1
Spiromesifen Insecticide GC-MS1; LC-MS/MS2
Spirotetramat Insecticide LC/LC-MS/MS2
Trifloxystrobin Fungicide GC-NPD1; GC-MS/MS2; LC-MS/MS2
Carbofuran
Diazinon
Malathion
Pesticide Use Residue Analytical Methods (RAM) Environmental
Protection Agency (EPA)1 or Literature2
10© 2017 The Cannabis Chemist ™2/15/2018
Compliance testing under OMMP
Potency THC, including THC acid CBD, including CBD acid CBN
Pesticides 20+ components
Mold/Mildew
% Moisture
HPLC MS/MSLC – QQQ
11© 2017 The Cannabis Chemist ™2/15/2018
LC-MS pesticidesAbamectin(Avermectins B1a and B1bBifenazateBifenthrin(synthetic pyrethroid)Cyfluthrin
(synthetic pyrethroid)
Daminozide (Alar)
Etoxazole
Fenoxycarb
ImazalilImidaclopridMyclobutanilPaclobutrazol PyrethrinsSpinosad Spiromesifen Spirotetramat Trifloxystrobin
CarbofuranDiazinonMalathion
12© 2017 The Cannabis Chemist ™2/15/2018
Measure 91 2016 - Oregon
420 dispensaries
50,000 growers
36,000 sites
34 labs
*Source: Oregon.gov
In order to implement Measure 91, the Oregon Liquor Control Commission
is required to establish a Rules Advisory Committee (RAC). The RAC is
charged with assisting in development of the fiscal impact statement and
helping the Commission develop the set of rules necessary to implement the
provisions of Measure 91.
Due to the complexity of the issues surrounding Measure 91, the OLCC will
establish technical committees of subject matter experts who will assist the
RAC and the OLCC in fully understanding the impact that the draft rules
will have upon specific areas of the regulated marijuana market.
Two Technical Committees; Licensing, Compliance & Enforcement, and
Labs will be ongoing to educate and inform the RAC and OLCC staff on
issues and topics in the recreational market.
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Unique sources of contamination
Products for ornamental use ONLY Roses have similar pests and bloom nutrients
Nutrient supplements Non labeled additives
CBD from Hemp Pesticides not allowed in US
Cross-contamination Extraction (CO2, butane)
Soils; persistent (Myclobutanil, etc.)
Applicators “Mfr opinion”
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Diversion – Cole Memo
Vice; like tobacco or alcohol
Price point for analytical
© 2017 The Cannabis Chemist ™ 152/15/2018
Venn diagrams to a practical path for safe cannabis
QuEChERs method has limitations
Electrospray Ionization (ESI) has limitations
HPLC required for many analytes
GC preferred for a few analytes
Certified cannabis labs; n = 0 Aug 2016
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“Action Levels”
Levels at which the labs are to be certified
FIFRA is still law:
10ppb limit
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No chemicals “approved” for use on cannabis
Tandem Quad is often the best solution…
Provides quantitative screening for known compounds for which there is a standard available
…but not always
Precise quantitative screening can be limited by:• Limited standard availability or elevated costs of uncommon compounds
• Infrequent occurrence of some compounds – why calibrate?
• Multiple matrix calibrations require time and resource
• ESI Bias for HPLC, Broad coverage via GC
© 2017 The Cannabis Chemist ™ 182/15/2018
Electrospray bias
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100%
+/- 3x
no cleanup
0
5
10
15
20
25
30
AO
AC
Qu
EC
hE
RS
/no
cle
an
up
Un
bu
ffe
red
Qu
EC
hE
RS
/no
cle
an
up
Ace
ton
itri
le (
FA
) /n
o c
lea
nu
p
No
n-v
ola
tile
ma
teri
al
(mg
/mL
)Gravimetric Evaluation
Pass throughcleanup
dispersiveSPE
cleanup
Different sample preparations
Thanks to Julie Kowalski
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5
Acetonitrile (0.1 % formic acid) no cleanup
AOAC QuEChERSPSA 50, C18 50, GCB 7.5 dSPE
AOAC QuEChERSPSA 100, C18 100, Chlorofiltr 50 dSPE
Acetonitrile (0.1 % formic acid) EMR Lipid blend
Acetonitrile (0.1 % formic acid)/ UCT pass through 2
Volatile Analysis (GC-MS)1g/10mL
1:20 dilution
Retention
time (min)
Pesticide Recovery Check
0
10
20
30
40
50
60
70
80
90
EMR Lipid blend
UCT pass through 2
69% and below 70% to 120%
% compounds in recovery ranges
30
12
70
88
% c
om
po
un
ds
QuECHERS?
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dSPE LC protocol
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dSPE GC protocols
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QuECHERS?
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QuECHERS?
Technology shift
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2016 – LC TQ $300k (refurbished) 2018 – LC TQ $600k+GC TQ $250k
new language/disciplineQuEChERs targeted cleanup
high dilution
improved technologyTQ TQ
(LC-ESI GC-EI)(ESI APCI)
New cost of entry
Importance of Food SafetyMarket Landscape and Dynamics
Brand ProtectionGlobalizationPopulation growth
Pesticides analysis represents $400M of the total Food Safety market with a project growth of 6%. Cannabis growth is well into double digits.
To meet expected demand in 2030, Food production needs to increase by 70%
Nationalization of cannabis supply chain means producers must accommodate many differing state regulations
ISO 17025 supporting cGMP
Negative Press can have lasting effect on Brand success in nascent markets
Prop 65 levies
Interstate cosmetics
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Compliance
© 2017 The Cannabis Chemist ™ 302/15/2018
Process Control + Feedback!
Extraction Process Butane
CO2
Ethanol
Fluorocarbon
Quality Control Dosing
Cannabinoids
Flavor/Fragrance
31© 2017 The Cannabis Chemist ™2/15/2018
Identification/QuantitationCombined Workflow by TOF
TOF is a technique which can combine benefits of quantitation with qualitative screening.
A full spectrum, accurate mass approach allows:
• Reliable identification of compounds, even without a standard
• Possibility to look for new compounds
• Retrospective data analysis
© 2017 The Cannabis Chemist ™ 322/15/2018
Enhanced Selectivity using Accurate Mass
EIC: 333 (± 0.5 amu) S/N (peak to peak): 2.5
EIC: 362 (± 0.5 amu) S/N (peak to peak): 8.9
EIC: 333.9826 (± 20 ppm) S/N (peak to peak): 21.8
EIC: 362.0139 (± 20 ppm) S/N (peak to peak): 23.5
TIC
Narrow mass extraction window improves s/n ratio and selectivity
Coumaphos 5 ng/mL spiked in blended food matrices extract
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Pesticide library
▪ Exact mass spectra for more than 850 pesticides and environmental contaminants
▪ Retention time locked screening methods
▪ Retention times help verify hits as another measure of confirmation in addition to exact mass spectra
PCDL = Personal Compound Database and Library© 2017 The Cannabis Chemist ™ 342/15/2018
Technology shift?
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2016 – LC TQ $300k (refurbished) 2018 – GC-TOF $600k+
new language/disciplineQQQ TOF
new technologyLC LC + GC
New cost of entry
New capabilitiesDiscovery/Isolations
MetabolomicsHuman -0mics
Conclusions Data tsunami
Broad based extraction and analysis
200 analytes @ 100ppb -> 700 analytes @ 10ppb
Targeted extractions and analyses
Cleanups
Platforms
Correct tools
Electronic filtering of data (workflow)
200 compounds x 50 samples/day x 3 ions = 30,000 data points
Retention time and ion ratios
Lower Limits of Quantitation (LOQ) approaching 10 ppb
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Future Scanning analyses for Tentatively Identified Compounds
Spectroscopy or Chromatography? Accurate Mass
LCxLC, GCxGC
Labelled internal standards for critical/problem analytes Class based or time based?
Bifenthrin
Imidacloprid
Accreditation; not just licensing Proficiency Test samples (in cannabis)
Primacy Labs/Methods AOAC
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Acknowledgements
Rodger Voelker
Rick Jordan/Steve Thun
Julie Kowalski
courageous public servants LC QQQ posse
2/15/2018 38© 2017 The Cannabis Chemist ™
Questions?
39© 2017 The Cannabis Chemist ™
(503) 403-8579
[email protected]/Facebook – The Science of Flower
2/15/2018