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©2017 ProVerde Laboratories, Inc.

Cannabis Contaminants:Practical Considerations vs Regulatory

Requirements

Christopher J. Hudalla, Ph. D.Founder/Chief Scientific Officer

ProVerde Laboratories, Inc.

Cambridge, MAApril 11th, 2017


Cannabis Contaminants

¡How do we cultivate?¡How do we process the plant?–Introduction of contaminants–Hazard Analysis and Critical Control Points (HACCP)

¡How do we measure and report contaminants?

¡How do we regulate?

Nature vs Human Nature


State by State Requirements

RequirementsbyState

Arizo

na

California

Colorado

Conn

ecticuit

Hawaii

Illinois

Maine

Massachusetts

Nevada

NewMexico

NewYork

Oregon

Rhod

eIsland

Washington

Cannabinoids - CN CN CN CN CN - CN CN CN CN CN - CN

Terpenes - TP - - - - - - TP - - - - -

HeavyMetals - - HM HM HM - - HM HM HM HM - - -

Pesticides - - PST PST PST PST - PST PST - PST PST - PST

Microbiological - MB+ MB+ MB+ MB+ MB - MB MB MB MB+ MB - MB

Mycotoxin - MY - MY MY MY - MY MY MY MY - - -

ResidualSolvents - VOC VOC - VOC VOC - VOC VOC VOC - VOC - -

Moisture - - - - MA - - - MA MA - MA - -

ForeignMaterial - FM - - - - - - FM - - - - -


Analytes and Analytical Techniques

Analytes Analytical Technique

Phyt

oche

mic

als Cannabinoids GC, HPLC, UPLC, CC (SFC), TLC, NIR

Terpenes GC-FID, GC/MS

Water (Residual Moisture) Gravimetric, Water Activity, pH

Genetics (Strain/Sex Testing) qPCR, Sequencing

Cont

amin

ants

Heavy Metals AA, ICP, ICP-MS, TXRF

Volatile Organic Compounds (VOCs) Headspace GC/FID, GC/MS

Mycotoxins ImmunoAffinity (IA) Assays, Strip Tests,LC/FLR, LC/MS

Microbiological Contaminants Cultures, qPCR, MPN, ELFA, ELISA

Pesticides/Plant Growth Regulators

LC/MS/MS, GC/MS/MS, GC/ECD, GC/VUV, ELISA


What’s in a unit?

Unit (Conc.) symbol 1 part in Example

Weight percent wt % 100 15 min out of a day

Parts per thousand ‰ 1,000 1.5 min out of a day

Parts per million ppm 1,000,000 32 seconds out of a year

Parts per billion ppb 1,000,000,000 3 seconds out of a century

“Do you guys do the ppm test?”

Anonymous


What’s in a unit?

¡ Lets make it MORE complicated:

Concentration vs Exposure Limitsmg/kg (ppm)µg/kg (ppb)

body weight per weekbody weight per day

From Nevada Regulations(now corrected)

Requires understanding a person’s body weight AND amount consumed to determine the

maximum allowable contaminants in a product

Mistakes are often propagated from one state to the next as regulators borrow

already crafted regulations


“Letters and Numbers Mean Something”

Technically speaking, 1 kilogram of soil can contain NO MORE than 400,000 kilograms of lead……


Focus on Contaminants

¡Pesticides, plant growth regulators, heavy metals, microbiological contaminants and process impurities

¡Consider route of administration– Inhalation, ingestion, sub-lingual, transdermal, rectal

¡Consider processes, matrices and actives– Extracts/Concentrates, tinctures, topicals, edibles, capsules/tablets,

vaporizer– Acid vs neutral form of cannabinoid


Pesticide Analysis

¡Pesticide guidance and limits vary widely by state–What pesticides are permitted for use?–What Pesticides to test for? Prohibited pesticides? Approved?–What levels are acceptable?

LimitsforPesticides(inppb)

Colorado 55pesticides?

Connecticut TolerancesandExemptionsforPesticideChemicalResiduesinFood- EPA(40CFR180(2014)

Nevada 74pesticides, LowestTolerancesfromEPACropGroup1910to10,000 ppb

Massachusetts 9pesticides,10ppb

Illinois TolerancesandExemptionsforPesticideChemicalResiduesinFood- EPA(40CFR180(2014)

California testing lab found pesticides in 84.3% of samples


Pesticide Analysis

Compound Nevada*(ppb)

Oregon(ppb)

Massachusetts(ppb)

Bifenthrin 50 200 10

Cyfluthrin 4,000 1,000 10

Etoxazole 7,000 200 10

Myclobutanil 9,000 200 10

Trifloxystrobin 11,000 200 10*BasedonEPATolerancesforCropGroup19andHops(SB447)

Disparity in state by state limits


Pesticides

Primary m/z 289>70

Confirmatory m/z 289>125


Pesticide Concentration with ExtractionConcentration of Myclobutanil

Extract Contaminationfrom contaminated flower

> 5 ppm

Flower: Myclobutanil Contamination


Heavy Metal Toxicity

¡Cannabis is well known to bioaccumulate toxic heavy metals from soil and environmental sources

¡Most toxicity data available is in reference to ingestion or environmental exposure of heavy metals

¡ Inhaled mainstream smoke is capable of transporting many substances through the mouth and throat and into the lungs to be absorbed or deposited

¡Toxicological research is needed to understand the implications of metal contaminant exposure on inhalation of smoked/vaped cannabis products

¡ Insufficient data to reliably set “safe” levels for these toxicants

¡Potential sources include: Water, growing medium, nutrients, processing equipment and hardware


Heavy Metals

¡Mercury, Cadmium, Arsenic, and Lead¡Metals are naturally occurring elements. Important to distinguish between ambient and contamination levels

¡Regulations vary dramatically by state

¡Whose limits are most appropriate?¡Test upstream potential sources? Periodic testing of products?

Limits (in ppb) Massachusetts Colorado Connecticut Hawaii

Cadmium (Cd) 200 4,100 900 4,000

Lead (Pb) 500 10,000 2,900 6,000

Arsenic (As) 200 10,000 1,400 10,000

Mercury (Hg) 100 2,000 2,900 2,000


Quantitative Elemental Analysis

¡ Inductively Coupled Plasma Mass Spectrometry (ICPMS)

¡ Detection to sub-ppb range

¡ Uses high frequency inductively coupled plasma as the ionization source for mass spectrometry


Heavy Metal Remediation

0

100

200

300

400

500

600

700

PPM

HeavyMetal

RawPlantMaterialExtractedResin

Lead (Pb) Arsenic (As) Cadmium (Cd) Mercury (Hg)

Pb Limit

As Limit Cd Limit

Hg Limit

*Limits as established by the Massachusetts Department of Public Health

High CBD Cannabis Sample


Sources of Contamination

¡Processing Equipment–Particulate residual from

equipment manufacturing

Fe, Cr, Ni(Stainless Steel)


Sources of Contamination

¡Vape Hardware–Heating elements–Aluminum, copper, nickel, and

chromium

Al, Cu, Ni, Cr


Residual Solvents

¡Which solvents are appropriate for use in production?–USP designation for class 1, class 2, and class 3 solvents–Are hydrocarbon extractions acceptable? If so, should a

minimum grade or purity of hydrocarbon be specified?

¡Which solvents should be tested for?¡What limits are appropriate?

–Limits that are too restrictive will result in significant product failures

¡What about testing for solvents used for cleaning?


Residual Solvents

¡Limits vary by state:

Residual Limits (ppm) Massachusetts Colorado Oregon Hawaii

Butane 12 5,000 5,000 800

Methanol 3,000 - 3,000 -

Ethanol 5,000 - 5,000 -

Hexane 290 290 290 10

Heptane 5,000 5,000 5,000 500

Benzene - 2 2 1

Toluene 890 890 890 1

Xylene 2,170 2,170 2,170 1


Residual Solvents

Time (min)0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

[mV]Vo

ltage

0

100

200

300

400

500 buta

ne

etha

nol

isopr

opan

ol

1,2-

dich

loro

etha

ne

1,326 ppm butaneMassachusetts Limit*: 12ppm

*Limits as established by the Massachusetts Department of Public Health


Microbiological Contaminants

¡Contamination of final products often due to other constituents or processes rather than cannabis raw material

¡Many production processes involve heating steps–Kills many microbial contaminants

–May not kill fungal spores

–May leave behind harmful residuals

¡ Importance of therapies with acidic cannabinoids requires production without typical heating steps (to avoid decarboxylation)–Special considerations should be made for these products



Traditional CultureTechniques

Most Probable Number(MPN) Enumeration

DNA Based- qPCR

- MicroArray



¡Most states use AHP guidelines as a basis for testing and limits.

¡Many states have added mycotoxin requirements¡Some states require additional speciation of contaminants¡What methodologies are most reliable?

¡ Yeast & Mold (YM: 10,000 CFU)¡ Enterobacteriaceae (EB: 1,000 CFU)¡ Total Coliform (CC: 1,000 CFU)¡ Total Aerobic (AC: 100,000 CFU)¡ Pathogenic Bacteria (None Detected)

– E. coli (O157)– Salmonella

Entero-bacteriaceae

Total Coliform

Fecal Coliform

E. coli

PathogenicE. coli

Total Aerobic


Important Considerations

¡State/Local Regulatory Agencies

¡ ISO Accreditation Bodies

–PJLA, A2LA, ACLASS, ANAB

¡ Instrument/Consumables Vendors

–Waters, Shimadzu, Agilent, Medicinal Genomics, Biomerieux, Restek, Cerilliant, Cayman, LGC

¡Established Organizations

–AOCS, AOAC, USP, ACS, AHPA, AHP, ASA, CSI, NIDA, FDA, ASTM

¡Collaboration between Cannabis Testing Labs

¡Standardized Testing Methodologies

¡Proficiency Testing


Summary

¡For many contaminants, toxicity is cumulative¡Cultivation and processing activities need to consider potential sources to minimize the potential for contamination (HACCP)

¡More work needs to be done to understand the toxicity of contaminants through various mechanisms of administration–Will enable regulators to set limits based on information rather

than fear

¡Regulatory considerations becoming increasingly important–Requirements and limits are continually evolving

¡Standardized methods and proficiency testing will help laboratories generate reliable data

¡Better communication is needed!


Acknowledgements

– John MacKay– Ronan Cleary– John Van Antwerp– Andy Aubin– Michael Young

– Sean Randall– Todd Medovich– Michael Parks– Max Lein

¡ ProVerde Colleagues– Jason Dunne– Lynsie Almeida– Matt Silva

– Pallavi Chhabra– John Mills– Timothy Wheeler

– Amanda Rigdon

– Kevin McKernan– Heather Ebling– Jessica Spangler


www.ProVerdeLabs.com

Download - Christopher J. Hudalla Cannabis Testing CannMed 2017 CJH · PDF file–Acid vs neutral form of cannabinoid ... ¡Some states require additional speciation of ... Christopher J. Hudalla

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