Microwave Digestion Analytical Chemistry Regulatory and Analytical

Technology for and Cannabis Challenges for Microbial

Measuring Heavy Metals Testing in Cannabis

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

Cannabis
Testing and
Calculating
Uncertainty

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4

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6 Table of Con
ntentts

CANNABIS SCIENCE AND TECHNOLOGY | VOL 1 • NO 3 w w w.CannabisScienceTech.com

CANNABIS ANALYSIS

1Cannabis
0 Welcome to
Analysis 22 38
Brian C. Smith
In this first installment, the author in-
troduces himself and the goals for 2 2 Microbial Testing in
Cannabis: Regulatory and
3ESI8andLC–MS/MS with
APCI Sources for
the column.
Analytical Challenges Meeting California Cannabis
CANNABIS VOICES Matthew A. Ward Pesticide and Mycotoxin
An outline of some of the current ma- Residue Regulatory
1Priscilla
4 A Queen’s Rise: How
Vilchis Became
jor challenges for both regulators and Requirements
laboratories regarding the microbial Avinash Dalmia, Erasmus Cudjoe,
Cannabis Industry Royalty testing of cannabis products. Toby Astill, Jacob Jalali,
Megan L’Heureux Jason P. Weisenseel, Feng Qin,
Priscilla Vilchis, owner and CEO of Molly Murphy, and Travis Ruthenberg
Premium Produce, discusses her ex- 3Digestion
0 Selecting Microwave
Technology for
How two different LC–MS/MS meth-
perience in the cannabis industry, ad- ods with ESI and APCI were used for
vice for newcomers, and more. Measuring Heavy Metals in low-level analysis of 72 pesticides
Cannabis Products
FEATURES Ryan Boyle and Eric Farrell CANNABIS CROSSROADS
A look at the basic principles of the
16 Cannabis Uncertainty major types of microwave digestion 5 1 The Cannabis
Science Conference
Brianna Cassidy and Cindy Orser technology as well as suggestions for
This article is intended to spark con- which might be the best approach Grows to New Levels
versations about measurement un- based on sample matrix, digestion Joshua Crossney
certainty that lead to the recognition efficiency, sample throughput, pro- Highlights from the 2018
and application of effective uncer- ductivity, and overall cost of analysis. Cannabis Science Conference
tainty budgets in the cannabis arena.
DEPARTMENTS

08 Cannabiis New
ws Fo
ocu
us

53 Applicattion No
otess

57 Prroduct Prrofile
es

58 Calll for Paperss

on the cover:
Ky Camacho/Shutterstock.com

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

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8 Cannabis Ne
ews Focus

Senators Push Bill to Legalize -Close-ups/macro shots of the flower are welcome
Medical Cannabis for Veterans -Photos of problem plants are also welcome (that is,
mold, contamination, etc.)
Two U.S. Senators introduced legislation on Wednes-
day, September 5, 2018, that would allow doctors at the Image size requirements are:
U.S. Department of Veterans Affairs (VA) to prescribe -Resolution – 300-dpi (or 2400 pixels x 3225 pixels)
medical cannabis to veterans in the 31 states that have -Color Mode – 8-bit CMYK
established medical-marijuana programs. The legisla- -Preferred File Format: TIFF (w/LZW or ZIP
tion was sponsored by Senators Bill Nelson (D-FL) and compression)
Brian Schatz (D-HI). -Acceptable File Format: JPEG (Quality 12/Progres-
According to a press release from Senator Nelson (1), sive 5 scans)
the bill would create a temporary, five-year safe harbor -Physical size of the cover will be 8” x 10.75”
protection for veterans who use medical cannabis. The For more information about the image requirements,
bill would also direct the VA to conduct research on the please visit: www.cannabissciencetech.com/article/
effects of medical cannabis on veterans who are in pain cannabis-call-cover-images-requirements.
and how prescribing cannabis to veterans can be used Submit your images online at: www.cannabissciencet-
to reduce opioid abuse. Veterans are currently twice as ech.com/submit-your-photo-cannabis-cover-image.
likely as civilians to die from opioid overdose.
“Federal law prohibits VA doctors from prescribing The Cannabis Science
or recommending medical marijuana to veterans,” Nel-
Conference Continues to Grow
son said in the press release. “This legislation will al-
low veterans in Florida and elsewhere the same access
to legitimately prescribed medication, just as any other The Cannabis Science Conference, which took place
patient in those 31 states would have.” August 28–29, 2018 in Portland, Oregon, saw another
For more information or to view a copy of the bill, year of record-breaking attendance and exhibitors.
please visit the press release link below (1). The conference drew in a large crowd with its high-
profile plenary speaker, Fran Drescher, who addressed
Reference a packed room on Tuesday. Drescher discussed the
1) https://www.billnelson.senate.gov/media- Cancer Schmancer nonprofit she runs and the move-
center/newsroom/senators-file-bill-allow-va- ment for cleaner living that is the basis of their mes-
prescribe-medical-marijuana-veterans sage. She also explained her involvement with the can-
nabis industry and how its use as a medicine has helped
Cannabis Call for Cover Images her heal.
Many attendees also came to hear world-renowned
scientists speak about their research such as Dr. Dedi
Are you an amateur photographer? Do you enjoy taking Meiri of Technion Israel Institute of Technology, Dr. Sue
photos of your cannabis plants, grow room, or facility? Sisley of Scottsdale Research Institute, Dr. Ethan Russo
Imagine your image on the cover of a magazine and of the International Cannabis and Cannabinoids Insti-
your name listed on the table of contents! tute (ICCI), and Dr. Bonni Goldstein of Canna Centers.
We are looking for submissions for 2019 covers of Other prominent leaders in the cannabis industry also
Cannabis Science and Technology. Specific image gave exciting talks: Tracy Ryan of CannaKids and Saving
guidelines include: Sophie; Dr. Jahan Marcu of the International Research
-No product photos (for example, specific instru- Center on Cannabis and Mental Health; Dr. Ralph Peroli
TOTEMART/SHUTTERSTOCK.COM

ments, brand names) of the National Research Council Canada; and Dr. Uma
-No people in the photos Dhanabalan of Uplifting Health & Wellness & Global
-No cannabis products in use (for example, no bongs, Health & Hygiene Solutions—just to name a few.
pipes, smoking joints) For more coverage of this year’s conference, please
-Artistic shots are welcome see “Cannabis Crossroads” on page 51 of this issue.

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

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10 Cann
nabis An
naly
ysis

Welcome to Cannabis Analysis

In this first installment, the author introduces himself and the goals for the column, including the
importance of establishing chemical analysis in the cannabis industry, helping those new to chemical
analysis with easy to understand explanations of important concepts, and serving as a repository for
new and interesting developments in cannabis analysis for those working in the field.

Brian C. Smith

I
am thrilled to be given the opportu- basic topics, introduce them to instru- you use mid-infrared spectroscopy to
nity by the editors of Cannabis Sci- mental techniques they might not be fa- determine THC in buds?” It took awhile
ence and Technology magazine to miliar with, and become a place to dis- for me to figure it out, but the answer is
write this regular column for them and cover new applications. yes (4). Since then I have been focused
for you. The purpose of this column, on the application of mid-infrared spec-
entitled “Cannabis Analysis,” is to dis- Who Am I? troscopy to the analysis of cannabis-
cuss analytical chemistry as it pertains What gives me the right to write a col- containing materials and have found
to the cannabis industry. Our indus- umn on cannabis analysis? Let me in- that it can determine cannabinoid and
try makes products that people use troduce myself. My experience in ana- terpene profiles in cannabis bud, trim,
for recreational and medicinal purpos- lytical chemistry goes back decades. I oils, extracts, shatter, resin, rosin, wax,
es, similar to the food, beverage, and earned my PhD in physical chemistry sugar, hashish, and kief (5–8). To com-
pharmaceutical industries. In those in- from Dartmouth College, and my the- mercialize my discoveries, I founded
dustries chemical analysis plays an im- sis research involved fundamental re- and am the Chief Technical Officer of
portant part in insuring the safety, ef- search using infrared (IR) spectrosco- Big Sur Scientific, manufacturers of the
ficacy, and profitability of products. It py. I have worked at several different BSS 2000 cannabis analyzer. It is a gen-
needs to perform the same role in the companies, including instrument com- eral purpose quantitative mid-infrared
cannabis industry. One of my goals for panies, as a research scientist and ap- spectrometer of unique design that is
this column is help analytical chemistry plications chemist. I continued my work small and portable.
serve that need. in IR spectroscopy, but also broadened As you can see, my degree, back-
Because of the history of illegality of my scope by becoming familiar with ground, and experience in analytical
cannabis and cannabis products, ana- other types of spectroscopy and the chemistry, combined with my research
lytical chemists did not become exten- many and varied types of chromatogra- in cannabis analysis, make me a good
sively involved in this industry until re- phy. For the largest part of my career, I candidate to write this column.
cently. This means there may be people ran an analytical chemistry training and
in the cannabis industry who perform consulting business. I have published a Cannabis is Medicine . . .
chemical analyses or interpret chemi- number of peer-reviewed scientific arti- Test it Like Medicine!
cal analysis results who might not have cles, and written three books on analyt- We might not want to think of it this
a technical background and might find ical chemistry (1–3). I am also an expe- way, but cannabis businesses are in
themselves struggling as a result. This rienced scientific columnist. For several the chemical processing industry. We
column is for you. I intend to explain the years now I have written a regular fea- can draw the analogy to whiskey mak-
basics of analytical chemistry as it ap- ture in Cannabis Science and Technol- ing. Barley is grown and bred to have
plies to cannabis. My goal then is if a ogy’s sister publication Spectroscopy a certain chemical composition, is har-
nontechnical person picks up an issue of magazine called “Infrared Spectral In- vested, ground up, and processed via
BLABLO101/SHUTTERSTOCK.COM

Cannabis Science and Technology they terpretation Workshop.” If you want to the chemical reaction of fermentation.
can read my columns to gain the under- learn how to interpret IR spectra, that This processed crop is then distilled to
standing they need to make sense of the column is a good place to start. give us the fluid that is bottled and pur-
technical content in the rest of the issue. My experience in the field of canna- chased by consumers. Now technically
However, this column is not just for bis analysis is more recent, but no less whiskey making is part of the food and
newbies. Seasoned scientists will bene- in depth. Shortly after recreational mar- beverage industry, but there is chemis-
fit from reading this column because it ijuana became legal in Colorado a col- try and chemical analysis involved every
will give them a refresher on important league of mine asked, “Hey Brian, can step of the way.

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

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12 Cann
nabis An
naly
ysis

What I just described is similar to how a cannabis distillate understand what goes into your process, what your process
is made. A crop is grown, cannabis, to have a certain chemi- does chemically, and monitor and control that process? You
cal composition, in this case a specific cannabinoid or terpene can’t. Thus, in this column I will be a passionate advocate for
profile. The crop is harvested, ground up, and extracted with increased use of chemical analysis in the cannabis industry
a solvent to make an oil. This oil can then be distilled to make (stepping off the soapbox now).
a cannabis distillate. Chemistry and chemical operations are
present throughout this process. Summary
With the U.S. Food and Drug Administration (FDA) finally In conclusion then, this column will serve multiple purposes. It
on record (9) that a cannabinoid (cannabidiol [CBD]) has a le- will introduce the basics of analytical chemistry to those new
gitimate medical use, even the harshest skeptics must now to the field, it will give analytical chemists the opportunity to
admit that medicinal cannabis is real. That means the canna- refresh and renew their knowledge, and it will advocate for
bis industry is not only a chemical processing industry, but we increased use of chemical analysis in the cannabis industry. I
are in the business of making medicine; that is, we are in the look forward to having many of you as regular readers.
pharmaceutical business. Since cannabis is medicine it should
be tested like medicine! Which means we as an industry have References
a lot to learn from the pharmaceutical industry in terms of how 1) B.C. Smith, Fundamentals of Fourier Transform Infrared
to process and test medicines. Spectroscopy (CRC Press, Boca Raton, Florida, 2011).
Drug companies practice what I call “cradle-to-grave”
2) B.C. Smith, Infrared Spectral Interpretation: A Systematic
chemical analysis. Raw materials coming in the door are an- Approach (CRC Press, Boca Raton, Florida, 1998).
alyzed for identity, potency, and purity. At all points during
3) B.C. Smith, Quantitative Spectroscopy: Theory and
the synthesis, blending, and processing of medicines meas- Practice (Elsevier, Boston, Massachusetts, 2002).
urements are made to ensure process quality, optimization,
4) B.C. Smith, M.A. Lewis, and J. Mendez, “Optimization
and product quality. Final product is tested before it goes out
of Cannabis Grows Using Fourier Transform Mid-
the door to be sure it meets specifications and meets the le- Infrared Spectroscopy,” PerkinElmer Inc. Application
gal requirement that the medicine is safe, effective, and la- Note, Waltham, Massachusetts, 2016.
beled properly for its intended use. 5) B.C. Smith, Terpenes & Testing Magazine,
I believe that the cannabis industry needs to adopt a cra- Nov/Dec(6), 48–51 (2017).
dle-to-grave testing model like the pharmaceutical industry
6) B.C. Smith, Terpenes & Testing Magazine,
for several reasons. First, we have a moral obligation to make Jan/Feb(7), 34–40 (2018).
sure cannabis medicines are safe and effective, which can
7) B.C. Smith and J. Strull, “Determination of
only be done through testing. Second, to increase the legality Cannabinoid and Terpene Profiles in Cannabis Bud
of cannabis the industry must do all it can in its power to pro- and Trim by Mid-Infrared Spectroscopy,” Cannabis
tect the health and safety of our customers. Cradle-to-grave Science and Technology, in preparation.
testing not only helps ensure health and safety, but will help 8) Big Sur Scientific website: www.bigsurscientific.com.
us make our case for increased legalization. Third, we have a
9) United Stated Food and Drug Association website:
legal obligation to test our products. The legal requirements https://www.fda.gov/newsevents/newsroom/
for testing may vary by jurisdiction, but the arrow of time in pressannouncements/ucm611046.htm.
my opinion is pointing toward increased regulation in all juris-
dictions. The industry might as well be ahead of the curve by
About the Columnist
performing increased testing now. Finally, it makes economic
sense. The pharmaceutical industry is required to test by the Brian C. Smith, PhD,
is Founder, CEO, and Chief Technical Officer of
FDA, but it performs many more tests than required because
Big Sur Scientific in Capitola, California. Dr. Smith
BLABLO101/SHUTTERSTOCK.COM

it helps increase their profitability. Analytical chemistry helps has more than 40 years of experience as an in-
them not pay for inferior material, optimize processes while dustrial analytical chemist having worked for such
companies as Xerox, IBM, Waters Associates,
minimizing cost and risk, and avoid lawsuits by making sure
and Princeton Instruments. For 20 years he ran Spectros
the final product meets label claims and is not harmful. Associates, an analytical chemistry training and consulting
It has been my observation that in many cannabis busi- firm where he taught thousands of people around the world
how to improve their chemical analyses. Dr. Smith has written
nesses the only time testing is done is on the final product.
three books on infrared spectroscopy, and earned his PhD in
How can you make a safe and effective medicine, comport physical chemistry from Dartmouth College.
with regulations, and increase your profitability if you don’t

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

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14 Cannabis Voice
es

A Queen’s Rise: How Priscilla Vilchis

Became Cannabis Industry Royalty
Priscilla Vilchis, owner and CEO of Premium Produce, was the first licensed female minority in L.A.
County, California, and the youngest female minority to be licensed in Nevada. She recently spoke
to us about her experience in the cannabis industry, advice for newcomers, and more.

Megan L’Heureux

P
riscilla Vilchis, owner and CEO industry initially, and it is my goal to an incredibly detailed business plan
of Premium Produce, a medi- use my experience in the healthcare outlining everything from sales track-
cal and recreational cultivation industry to generate reimbursements ing to security measures and demon-
and processing company with op- for medical cannabis. strate that we had the financial back-
erations in Las Vegas, Nevada, and I am flattered that people refer to ing to execute our plan.
Lynwood, California, and addition- me as the “Queen of Cannabis.” It is The California applications are
al delivery and distribution licenses truly motivating. I am humbled. also very rigorous, but the pro-
in California, has quickly become a cess is a bit different. Unlike in Ne-
major player in the cannabis indus- What made you want to start vada where the cannabis industry
try. Vilchis was the first licensed fe- cannabis businesses in California was brand new, cannabis businesses
male minority in L.A. County and the and Nevada? have been operating in a legal gray
youngest female minority to be li- California and Nevada have been area in California for many years. The
censed in Nevada—both very hard- leaders of the legalization movement. state began continuously accepting
won achievements. Here Vilchis dis- I entered this business in Nevada be- applications on January 1, 2018 and
cusses the process she went through cause of the comprehensive, com- has created “temporary licenses”
to start her businesses, advice for mon-sense regulations. I expanded to bridge the gap and allow canna-
people starting out, and hopes for operations to my home state of Cal- bis businesses with local authoriza-
the future. ifornia when the state implemented tion to stay in operation while the de-
a regulatory framework for the indus- tailed annual license applications are
You’ve been nicknamed the try. I am passionate about bringing under review. Another thing that is
“Queen of the Desert” or “Queen the law into line with reality and creat- different about California are higher
of Cannabis.” Can you tell us how ing safe and effective cannabis prod- application or license fees and taxes
you got so involved in the cannabis ucts for medical marijuana patients
industry? How do you feel about and responsible recreational users.
those nicknames? The market demand in these states
I got my start in the healthcare in- is huge. Los Angeles is predicted to
dustry managing several physicians be the biggest cannabis market in the
throughout Southern California and world, and Las Vegas is the ultimate
rapidly expanded to different states. cannabis tourist destination.
I learned a lot about the use as well as
the abuse of prescription drugs and What was the process like to get
became interested in alternatives to your licenses? How did it differ from
opioid-based medications. California to Nevada?
I discovered that cannabis and can- The medical marijuana establish-
nabinoids have a wide range of thera- ment application process in Nevada
peutic potential. I believe that the use was extremely rigorous and com-
of medical cannabis will result in sav- petitive. It was only open for a brief
ings to health insurance systems. This period of time, and drew more than Figure 1: Priscilla Vilchis at her grow
facility in Nevada.
is what brought me to the cannabis 500 applications. We had to include

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Cannabis Voiices 15

on cannabis operations. These are that is extremely difficult to navi- would protect the growing marijua-
very high barriers to entry, and many gate. I strongly believe that the laws na industry. States that have already
established cannabis operators are will change and things will get bet- legalized cannabis are also taking
struggling to make the transition into ter for our industry in Nevada, Cali- a stand. I am looking forward to a
the legal market. fornia, and across the United States. great banking system, which would
Federal lawmakers have proposed lead to more safety and transparen-
What is the most difficult aspect of numerous pieces of legislation that cy in the industry. ◾
running a cultivation and processing
business? Are there specific tools or
technology that are a big help? Informatics for Your Lab – LIMS • ELN • LES
There are an extraordinary amount
of construction and design costs
when building a cannabis opera-
tion. In addition to all the fees paid
for licensing, the build out costs are
substantial.
Our Nevada facility features a cus-
tom-made Link4 environmental con-
trol system, which cost more than $1
million to purchase and install. It con-
trols temperature, humidity, and air-
flow, and monitors irrigation to warn
staff when parts-per-million levels
are too high and when filters need to
be changed. It also plugs into light
sensors to alert the team when a
light bulb needs to be changed be-
fore grow room light levels become
uneven. The automation is a big help
and worth every penny.

What advice would you offer to

others looking to start a cultivation
and processing business?
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VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

16 Feature

Cannabis Uncertainty
How certain can you be that every bud within a container labeled 9.25% ∆9-tetrahydrocannabinolic
acid (THCA) is exactly 9.25% THCA? Wouldn’t you like to know? The uncertainty of the measurement
of THCA must be accounted for. Uncertainty, in an analytical context, refers to the range around a
reported result within which the true value can be expected at a certain probability. Intensively grown
cannabis plants are highly heterogeneous. Cannabis heterogeneity is contributed genetically from
decades of poorly documented hybridization experiments and compositionally from environmental
factors. These variables, among others, affect the distribution of key active ingredients and are
also likely to cause imbalanced distributions of contaminants such as heavy metals and pesticides.
Therefore, no matter how accurate and precise a testing laboratory is in measuring cannabinoids
or contaminants, the resulting uncertainty introduced by the plant plays a large role. Independent
testing laboratories servicing the cannabis industry contend with this inherent cannabis uncertainty on
a daily basis while complying with dynamic state-by-state testing mandates and client expectations.
Here we illustrate the uncertainty contributed by cannabis heterogeneity and discuss the uncertainty
contributed by laboratory measurement. We propose an accurate and transparent alternative method
of relaying active ingredient content, and reference regulatory guidance for handling measurement
uncertainty while quantifying and reporting contaminants. A specific example of how measurement
uncertainty is involved in determining the pass or fail status of a product in regard to maximum
residue limits is also provided and discussed. This article is intended to spark and aid conversations
about measurement uncertainty that lead to the recognition and application of effective uncertainty
budgets in the cannabis arena.

Brianna Cassidy and Cindy Orser

T
his manuscript describes that characterizes the dispersion of consumables from different lots,
“measurement uncertainty” values that could reasonably be at- and so forth. The maximum varia-
(MU) from the perspective of tributed to the measurand” (1). In bility that the measurement meth-
two International Organization for the previous issue of Cannabis Sci- od may encounter, within bounds
Standardization (ISO)-17025 accred- ence and Technology, Patricia At- defined in the standard operating
ited independent testing laborato- kins provided an empirical descrip- procedure, should be explored and
ries that analyze cannabis products tion of MU (2). This article provides included during replicate analysis.
for potency, terpenes, homogene- a link between empirical discus- It can be difficult to identify and in-
ity, microbials, mycotoxins, heavy sions on result uncertainty and their clude all factors of uncertainty pre-
metals, residual solvents, and pesti- direct application to measurements sent in a measurement. Therefore,
cides. Focus is placed on measure- in cannabis. we recommend first describing and
ment uncertainties when analyzing MU can simply be defined as the reducing the MU contributed by the
cannabis inflorescence, since can- “give or take” of a measurement. laboratory method, then using the
nabis extracts and infused products This uncertainty is experienced in optimized method to determine
are more homogeneous (if prop- everyday life whenever a measure- the MU added by the sample. We
erly formulated). Under the inter- ment is taken (how hot, how tall, discuss the laboratory and sample
national standard, ISO-17025:2017, or how heavy something is). Fac- contributions to MU separately, and
accreditation organizations are re- tors that contribute to MU should refer to the combined MU, along
questing that laboratories make be identified and reduced when with any adopted expansion, as the
available MU associated with labo- possible. Uncertainty can be de- uncertainty budget.
ratory methods. The official defini- termined by measuring the same Some regulatory guidance advis-
tion of MU is “a parameter associat- sample prepared by different an- es describing the uncertainty con-
ed with the result of a measurement alysts, on different days, using tributed by each identified factor,

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 17

and then combining the uncertain-

ties (by calculating the square root
18
of the sum of the square of each
16
identified uncertainty). This method
14
requires separate calculations for

Percent THC
12
each matrix type (flower, solvent ex-
10
tract, nonsolvent extract, and every
8
different type of cannabis infused 6
product) at each level of expect- 4
ed concentration for every analyte 2
measured. The number of experi- 0
ments required to accomplish such 1 2 3 4 5 6
a task is daunting. In a field where Plant
products and testing regulations
are everchanging, more plausible
ways to determine uncertainty use Figure 1: Percent THC in six randomly selected flower samples from each of six plants
within the same batch. Adapted with permission from reference 6.
means of accounting for unexplored
variability, such as a “k factor.” A k
factor is a multiplier applied to the MU calculation and provides more expanded uncertainty can be found
found or calculated uncertainty that confidence that the true value ex- in guidance documents that pro-
widens the MU range. It is meant to ists within the reported range (95% vide predetermined expanded un-
account for aspects of uncertainty confidence if a k factor of 2 is used) certainties for a wide range of ana-
that may have been left out of the (3). Another conservative form of lyte concentrations (4).
18 Feature

Little regulatory guidance ex- Zealand police officers purchased a remedy, but this approach would
ists on the topic of MU and how it plants from an illegal grower. Cut- just give a more accurate estimate
is to be considered when determin- tings from a single plant were used of the average value and would not
ing the pass or fail status of an an- to grow six clone plants. The vari- account for the expected range in
alytical result. If a state regulation ability of cannabinoid content be- values from bud-to-bud. Since ho-
limits a residue to a concentration tween and within plants is evident. mogenizing cannabis before sam-
of 10 parts per billion (ppb), and a Though some of the variability may pling and sale is not a lucrative op-
sample is found to contain 11 ppb be attributed to the inexperience tion, a more representative way of
of that residue, is that sample out- of the growers, it is not realistic to reporting cannabinoid content has
side of compliance? What if the MU assume that even the most distin- been suggested, which is to report
is ±50% at the 10 ppb level? The Eu- guished growers produce batches a range rather than an absolute val-
ropean Commission provides clear of flower where all buds are equal ue (7). Determining the range of an-
guidance in this situation regard- in potency. Expecting all buds with- alyte concentrations within a batch
ing pesticides (5), but state-by- in a container to be of the same po- of cannabis flower would be a rig-
state regulations for cannabis test- tency is like expecting all grapes orous process, requiring enough
ing do not. This article provides a within a bag to be of the same ripe- separately tested buds to describe
glimpse into the intricacies of can- ness. Similarly, contaminant levels the span of concentrations present
nabis testing uncertainty. We pro- are likely to be heterogeneously within a defined probability. The
pose implementation of expanded distributed and stored by canna- point is, if homogenization is not an
uncertainties, which should be con- bis. However, to our knowledge, no option, the direct alternative is to
sidered by regulators when setting studies are available that describe quantify the inhomogeneity. Labe-
maximum residue limits (MRLs), and the nature of contaminants in can- ling finished cannabis product with
a transparent method of relaying nabis. The inherent inhomogene- a range of active ingredient con-
MU of active ingredients to canna- ity of cannabis flower poses a se- tent, instead of an absolute value,
bis consumers. rious challenge that needs to be would temper cultivator expecta-
addressed for cannabis to continue tion and allow consumers to make
Sources of Uncertainty gaining integrity in the advancing, more accurate and informed dosing
Contribution to Uncertainty evidence-based medicine trajecto- decisions.
From Cannabis Inhomogeneity ry of the cannabis industry. Current laboratory practices are
We all have seen and know what in- One of the most effective, yet in- limited by the amount of product
tensive agricultural monoculture dustrially unappealing, ways to mit- submitted for testing, and there is
looks like, think amber waves of igate cannabis inhomogeneity is little encouragement to request
grain or an ocean of corn plants, all to simply grind and mix (homoge- replicate testing of the same batch
uniform and genetically homogene- nize) the entire batch of flower be- to explore the variability of cannab-
ous. However, that same conclusion fore testing and dispensing. Buds inoid or contaminant concentration
does not apply to intensively mon- at the extremes of cannabinoid within and between plants. When a
ocultured cannabis. Potency is de- and contaminant concentrations sample is submitted, it is homoge-
pendent on flower maturity, and it would dilute one another, result- nized by the laboratory before test-
is unlikely that all flowers within a ing in a more predictable product. ing. This step minimizes the chance
plant are equal in cannabinoid con- Not to mention the sample taken of measuring subsamples that are
tent at a given time. It is even less of the homogenate would be much extreme in concentration or con-
likely that all flowers within a batch more representative of the sample tamination, which gives a result that
of plants are equal in maturity at the than even the most rigorous analyt- will more likely represent the aver-
time of harvest. Within-plant varia- ical sampling of intact buds. Unfor- age of the batch. Testing laborato-
bility and plant-to-plant variability tunately, ground cannabis flower is ries cannot completely control the
regarding potency in cannabis has not currently an appealing product, uncertainty added by sample het-
been previously demonstrated (6). so homogenizing cannabis would erogeneity that exists before their
The results shown in Figure 1 reduce marketability. interaction with the sample. Ne-
were obtained from an experi- Keen analysts may suggest tak- vada regulation requires laborato-
mental indoor hydroponic can- ing a larger, more representative ry staff to visit grows to analytical-
nabis grow study in which New sample of the batch for testing as ly collect representative samples

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 19

for analysis, but they are still limit-

Table I: Acceptable recovery, specified by AOAC, as a function of analyte concentration (4)
ed on the amount of product avail-
able for sampling, and sample size Concentration Recovery Limits (%)

is directly correlated with the de- 100% 98–101

gree to which the sample repre- 10% 95–102
sents the batch. Massachusetts 1% 92–105
regulation holds the cultivators of
0.1% 90–108
cannabis products responsible for
0.01% 85–110
sample selection, which further
10 μg/g (ppm) 80–115
limits the capability of the labora-
tory to describe sample variabili- 1 μg/g 75–120
ty and sample representativeness. 10 μg/kg (ppb) 70–125
For these reasons, we recommend
identifying factors in MU of the lab-
Table II: The AOAC defines acceptable values for repeatability to be 0.5–2 times the
oratory methods apart from sample
values included in this table (4)
variability by using replicates of ho-
Concentration Repeatability (RSDr), %
mogenized sample. Separating fac-
tors of MU contributed by the lab- 100% 1
oratory from factors contributed by 10% 1.5
the sample mitigates complications 1% 2
related to sampling, and properly 0.1% 3
represents the accuracy and preci-
0.01% 4
sion of the laboratory.
10 μg/g (ppm) 6

Contribution to Uncertainty 1 μg/g 8

From Testing Laboratories 10 μg/kg (ppb) 15

Measurement uncertainty applies to
all forms of quantitative testing (4).
Table III: AOAC acceptable values for reproducibility are 0.5–2 times the values included
The true value of a measurement is in this table (4)
an ideal that in principle is unknown.
Concentration Reproducibility (RSDR), %
Common sources of uncertainty that
100% 2
should be considered in the meas-
urement method include, but are not 10% 3
limited to, the following parameters: 1% 4
repeatability, reproducibility, recov- 0.1% 6
ery, stability, and bias. Sources of 0.01% 8
uncertainty should be identified for
10 μg/g (ppm) 11
each analyte in each matrix type at
1 μg/g (ppm) 16
each expected concentration level.
10 μg/kg (ppb) 32
Cannabis laboratories analyze any-
thing from honey extracts to canna-
bis-infused suppositories, and each regulate more than 100 analytes in repeatability, and reproducibility for
matrix brings a different array of pos- cannabis. Massachusetts has 91, Ne- a wide range of analyte concentra-
sible uncertainty-impacting charac- vada has 63, and these numbers will tions (see Tables I–III) (4).
teristics to the table. Concentrations likely increase. Accounting for all fac- Acceptable recovery ranges have
of cannabinoids range from a frac- tors of uncertainty under these cir- been defined differently in other
tion of a percentage to almost 100% cumstances is an arduous task. regulatory guidance. The AOAC ref-
of product weight and contaminant We recommend starting with guid- erence the 70–120% recovery criteri-
levels are required to be measured ance from the Association of Analyt- an set by the U.S. Food and Drug Ad-
at parts-per-million (ppm) to parts- ical Communities (AOAC), who sug- ministration for drug residues at the
per-billion (ppb) levels. Some states gest ranges of acceptable recovery, 10 ppb level and the 50–150% critrian

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

20 Feature

set by the U.S. Department of Agri- of the results is expressed as percent change a variable in the measure-
culture pesticide residue proficiency relative standard deviation (%RSD), ment process.
study, but ultimately advise that re- which is the standard deviation di- The reproducibility standard de-
coveries outside of 60–110% be in- vided by the mean, multiplied by viation or performance character-
vesitgated and improved (4). Low 100. As the concentration of a giv- istic obtained in a round robin is a
recoveries are commonly because en analyte decreases, the allowed suitable measurement of uncertain-
of inefficient extraction methods, %RSD increases (see Table II). ty because it already reflects the
while recoveries greater than 100% The ability of a laboratory meth- methodological effects due to the
are possible because of matrix in- od to demonstrate the agreement of different ways laboratories operate.
terferences in chromatography and results from the same sample over a Between-laboratory results are dif-
ion enhancement in mass spectrom- given time span is a fairly strong in- ficult to quantitate because of the
etry. Cannabinoid recovery presents dication of the quality of the labo- competitive nature between labora-
an unusually difficult situation for re- ratory. Clients may test laboratory tories in the same state, and because
covery experiments. Most recovery repeatability by thoroughly homog- cannabis is federally illegal and can-
experiments are carried out by add- enizing a sample, splitting the sam- not cross state lines. However, data
ing a known amount of standard to a ple into several sealed containers la- resulting from the aforementioned
blank or representative matrix, then beled with different identifiers, then method can be used by clients to as-
dividing the analytical result by the sending the samples to laboratories sess laboratory quality and to deter-
theoretical result. No known blank for testing. The results can be used mine a reproducibility result if more
or representative matrix has been to calculate the laboratory %RSD. If than one laboratory was included.
established for cannabis. Further- the client is interested in the labora- Simply calculate the %RSD for all re-
more, even if an appropriate blank tory repeatability over a longer pe- sults from different laboratories for
matrix were agreed upon, cannab- riod of time, the samples should be the same sample combined into one
inoid standards are not available in submitted days apart. If the sample dataset. If a laboratory has already
concentrations above 1 mg/mL. Low- was not thoroughly homogenized determined the MU for the method
concentration cannabinoid stand- (solid samples ground to a powder in question, then the results of the
ards limit recovery investigations to and liquid samples mixed vigorous- interlaboratory comparisons can be
the lowest fraction of the range of ly), repeatability results may not be useful in checking this MU.
expected cannabinoid concentra- representative of the laboratory. Fur- Recovery, repeatability, and repro-
tions found in cannabis. As Table I thermore, if sample degradation oc- ducibility are just a chip off the ice-
demonstrates, recovery is expected curs from the time the first sample berg in determining laboratory MU.
to be dependent on concentration, is analyzed to the last, results will Additional types of MU likely enter
so the spiking method is not capable not be representative of the labo- into a quantitative result and con-
of describing the expected recovery ratory. These two factors should be tribute to the uncertainty budget.
for a majority of cannabis samples. kept in mind during all repeatability Many resources exist that provide
AOAC Official Methods of Analysis, experiments. guidance on measuring additional
Appendix K, suggests that samples Reproducibility is similar to re- factors of MU such as: Eurachem (8),
of this nature be evaluated for recov- peatability, but usually includes a Nordtest (9), Eurolab (10), and Codex
ery by re-extraction after the original wider span of purposefully changed CAC/GL 59-2006 (11).
extraction (4). Recovery would then variables and therefore has larger ac-
be calculated by dividing the first ex- ceptable uncertainties (see Table III). Conclusion
traction result by the sum of the first, Here we have the classic situation of The need for cannabis regulato-
second, and third extraction. This a round-robin or blinded proficien- ry guidance to recognize and ac-
method allows assessment of recov- cy test across numerous laborato- count for uncertainty in cannabis
ery of cannabinoids in cannabis at all ries where reproducibility conditions measurements has been made ap-
possible concentrations and does include the different methods, dif- parent. This article is not intend-
not yield recoveries more than 100%. ferent laboratories, different oper- ed to provide clear solutions to the
Repeatability is examined by us- ators, and different equipment and complex issues at hand, but rath-
ing the same method, laborato- instrumentation. What makes re- er to demonstrate the urgent need
ry, equipment, and instrumentation producibility different from repeat- for discussion on the topic and to
within a given time span. Variability ability is the need to purposefully demonstrate the importance that

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 Featture 21

for Single-Laboratory Validation

uncertainties bring to the cannabis x – U = 2.2 – 1.1 (= 50% of 2.2) of Chemical Methods for Dietary
industry. = 1.1, which is > MRL. Supplements and Botanicals.
Uncertainty budgets are valua-
5) European Federation of National
ble diagnostic tools in the devel- This European Commission Guid- Associations of Measurement,
opment and optimization of meas- ance specifically states that it is to Testing and Analytical Laboratories,
urement procedures. Importantly, be used “to support compliance EUROLAB Technical Report
1/2006: Guide to the Evaluation
MU is a useful barometer for report- with, and specific implementation
of Measurement Uncertainty for
ing of both active ingredients, such of ISO/IEC 17025.” However, dis- Quantitative Test Results. http://
as Δ9 -tetrahydrocannabinolic acid cordance between regulation guid- www.eurolab.org/documents/
(THCA), and contaminants, such as ance is plentiful. Risk factors should EL_11_01_06_387%20Technical%20
report%20-%20Guide%20
myclobutanil, in the ongoing effort be used to determine acceptable
Measurement%20uncertainty.pdf.
of cannabis testing laboratories to criteria and MRLs should be decid-
provide more transparency for the ed on with chosen MU criteria in 6) G. Knight, S. Hansen, M. Connor,
H. Poulsen, C. McGovern,
cannabis end user. Without inclu- mind. Cannabis is a new product and J. Stacey, Forensic Sci.
sion of MU, the misconception that with widely unexplored potential. Int. 202(1–3), 36–44 (2010).
the average value is representative Knowledge of how contaminants
7) M. Sexton and J. Ziskind,
of each bud within the product will interact and persist in cannabis, “Sampling Cannabis for Analytical
prevail. Making uncertainty budg- along with toxicological studies Purposes,” BOTEC Analysis Corp.
ets available, and incorporating on the inhalation of contaminants, I-502 Project #430-1e, (2013).
them in laboratory reporting, would should be used to shape future un- 8) EURACHEM/CITAC Guide,
ultimately result in a more transpar- certainty budgets. Quantifying Uncertainty in
ent and reliable cannabis market. Both laboratories await to receive Analytical Measurement, 3rd
Edition, 2012, http://www.
Conclusive uncertainty budgets, further guidance on how their re- eurachem.org/images/stories/
and guidance on how uncertain- spective states will acknowledge guides/pdf/QUAM2012_P1.pdf.
ty budgets are to be considered in the role of uncertainty budgets in
9) NORDTEST Report TR 537:
determining sample MRL compli- the calculation of reportable results “Handbook for Calculation of
ance, have yet to be well defined in on certificates of analysis. In the Measurement Uncertainty in
the cannabis arena. European Com- meantime, Digipath Labs, CDX Ana- Environmental Laboratories,”
http://www.nordicinnovation.
mission Guidance provides a direct lytics, and a handful of other canna-
net/nordtestfiler/tec537.pdf,
example of how uncertainty budg- bis testing laboratories are letting 2nd edition, Espoo, 2004.
ets are to be considered in deter- both their respective states and
10) European Federation of National
mining sample pesticide MRL com- their clients know that MU budgets Associations of Measurement,
pliance (10): are available to view. Testing and Analytical Laboratories,
EUROLAB Technical Report
For official food control by References 1/2007: Measurement uncertainty
revised: alternative approaches to
regulatory authorities, com- 1) International Organization for uncertainty evaluation,
pliance with the MRL must Standardization, ISO 17025:2017 www.eurolab.org, Paris, 2007.
be checked by assuming that Handbook. http://www.eurolab.
org/documents/EUROLAB%20 11) Codex Alimentarius Commission,
the MRL is exceeded if the CAC/GL 59-2006 (Amendment
Handbook%20ISO%20IEC%20
measured value exceeds the 17025%202017.pdf. 1-2011) Guidelines on Estimation
MRL by more than the ex- of Uncertainty of Results, www.
2) P. Atkins, Cannabis Science and codexalimentarius.net/download/
panded uncertainty (x – U
Technology 1(2), 44–48 (2018). standards/10692/cxg_059e.
> MRL). With this decision pdf, Rome 2006 and 2011.
rule, the value of the meas- 3) “Guide to the Evaluation of
Measurement Uncertainty for
urand should be above the Quantitative Test Results,”
MRL with at least 97.5% con- EUROLAB Technical Report
fidence. Thus, the sample is 1/2006, www.eurolab.org. Brianna Cassidy, PhD, is with CDX
considered noncompliant if 4) AOAC official methods of analysis Analytics, in Salem, Massachusetts.
Cindy Orser, PhD, is with Digipath
x – U > MRL. [For example], in (2013) guidelines for dietary
Labs, in Las Vegas, Nevada. Direct cor-
case the MRL = 1, the result supplements and botanicals,
respondence to: [email protected]
Appendix K, AOAC Guidelines
x = 2.2 and U = 50%, then

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

22 Feature

Microbial Testing in Cannabis:

Regulatory and Analytical Challenges
Cannabis testing regulations are constantly evolving with the industry as it grows and gains
acceptance. Currently, microbial contaminant testing regulations vary on a state-by-state basis and
sometimes are lacking or inconsistent. As a result of the variability and continuous and sometimes
hasty changes, the testing of cannabis product for microbial contamination faces constant and
evolving challenges. Of the many current challenges facing regulators and laboratories regarding
both testing and product safety, the most glaring are varying sample sizes, impossibly short testing
deadlines, enumerable product types released to the market, a lack of standardized industry testing
conventions, and incongruities in testing requirements. These challenges amount to a sizable
regulatory and testing gap in the industry; a gap in which cannabis is tested for contaminants, but
the collected data can sometimes lack statistical validity or mislead to assumptions of product safety.
Despite these difficulties, numerous industries outside of cannabis have faced and solved similar
problems and can offer solutions directly applicable to the cannabis industry. This article outlines
some of the current major challenges for both regulators and laboratories regarding the microbial
testing of cannabis products. The article is further intended to provide information and resources to
aid laboratories and regulators in the eventual resolution of the highlighted difficulties.
Matthew A. Ward
GIROSCIENCE/SHUTTERSTOCK.COM

M
icrobial testing in cannabis has become stand- methods seem to be the only true constants. In gen-
ard practice and a requirement across most eral, states usually require an array of tests, including
states with legal markets. Although the regu- total yeast and mold or select mold species identifica-
lations vary from state to state, the general challeng- tion, Salmonella (<1 colony forming unit [CFU]/g), tox-
es facing microbiologists are more consistent. Rapid igenic E. coli (<1 CFU/g), total aerobic plate count, to-
regulatory changes and a lack of general consensus on tal enteric count, as well as other specific bacterial

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 Featture 23

contaminants. But very seldom do To add to the complexity, meth- Challenges to the Legitimacy
state regulations exactly match one odologies adopted in various mi- of Microbial Testing
another. New York, for example, re- crobial testing categories vary tre- “Is microbial testing really neces-
quires a vast array of testing not mendously and include older, tried sary?” That’s a question often raised
seen in other legal states (Clostridi- and true compendial methods in Colorado.
um spp., Streptococcus spp., Penicil- adopted from other industries (or At first glance, the answer should
lium spp, Aspergillus spp, and Mu- variations of compendial methods) be a resounding “yes!” but legitimate
cor spp), while Colorado requires that are run alongside innovative concerns are being raised about what
only three microbial tests (3,15). new methods which are less heav- is being tested for and why. Howev-
These inconsistencies as well as in- ily vetted. New methods generally er, a far less valid argument, but one
dustry lobbying have led to skepti- are faster and have better sensitiv- still faced by microbiologists and reg-
cism regarding microbial testing as ity, but often are not nearly as well ulators, is the question: “why test if
a legitimate need in the cannabis in- tested in application. Although the no one has gotten sick from . . . Sal-
dustry. However, depending on the challenges seem daunting, it is im- monella, E. coli, and so on?” While
product type and the end consum- portant to keep in mind that every it might be true that contamination
er for which the product is destined, fledgling industry has faced simi- of certain products by say, E. coli or
microbial testing is absolutely nec- lar challenges and, while cannabis Salmonella might be unlikely, “what
essary; but it is a valid concern that can, at times, appear as though it is if’s” must always play a role when an-
testing requirements are simply ap- completely new, it does have strik- alyzing the risks involved with prod-
plied to all matrix types regardless ing similarities to both the food and uct manufacture and distribution.
of the intended use or consumer. environmental industries. The cannabis industry is young, and

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VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

24 Feature

Table I: APHL regulatory guidelines

Preparation Definition USP Microbial Limits
Total aerobic microbial count < 103
Other raw materials
Total combined yeast and mold count < 102
and ingredients
Absence of E. coli in 10 g
Nutritional products Total aerobic microbial count < 103
with other highly Edibles Total combined yeast and mold count < 102
refined ingredients Absence of E. coli in 10 g
Rectal suppositories
Total aerobic microbial count < 103
Rectal use products For nonsterile products for pharmaceutical prep-
Total combined yeast and mold count < 102
arations and substances for pharmaceutical use
Total aerobic microbial count < 102
Ointments, creams, inserts, and so forth
Total combined yeast and mold count < 10
Vaginal use For nonsterile products for pharmaceutical prep-
Absence of Pseudomonas aeruginosa, Staphylo-
arations and substances for pharmaceutical use
coccus aureus, and Candida albicans in 1 g or 1 mL
Total aerobic microbial count < 102
For nonsterile products for pharmaceutical prep- Total combined yeast and mold count < 10
Transdermal patches
arations and substances for pharmaceutical use Absence of Pseudomonas aeruginosa,
Staphylococcus aureus
Total aerobic microbial count < 102
Oral mucosal, gingival,
For nonsterile products for pharmaceutical prep- Total combined yeast and mold count < 10
cutaneous, nasal, or
arations and substances for pharmaceutical use Absence of Pseudomonas aeruginosa,
auricular use
Staphylococcus aureus
Must meet the requirements of USP
Ophthalmic use
771 for Ophthalmic Preparations

historical outbreak data is extreme- criteria for marijuana bud as strict as important to take the proper time
ly limited, but the problems faced by <1 CFU/g of bile-tolerant Gram-neg- to vet these decisions and, if neces-
other manufacturing industries are ative bacteria as well as <1 CFU/g for sary, to contract with the proper con-
directly similar and it is never accept- total mold, which would have mandat- sultants who have experience making
able to base testing solely upon what ed a product impossible to produce. these types of risk-based evalua-
has happened in the past (12). To ad- Current regulations across the coun- tions. In general, products intended
dress the issue of determining appro- try are generally strict and some are for adult-use consumers might not re-
priate target contaminants in canna- arguably overbearing. At the same quire extreme levels of testing; med-
bis products, a few basic questions time, it is almost always better to err ical products should, however, un-
should be considered (12): on the side of caution and if a product dergo extremely stringent levels of
• Who is the product destined for? could be destined for immunocom- testing to protect immunocompro-
• What is the likelihood of promised individuals (the very old, mised patients (6,11,12). For example,
contamination in the product? sick, or very young), then strict testing direct inhalation of 102 CFU/g yeast
• What impact would there be if requirements are indeed necessary and mold might not be a problem for
the product was contaminated? (5,12). To aid regulators in promul- an immunocompetent individual, but
• Are there process controls or steps gating rules that have precedent, the for someone with a debilitating dis-
in manufacturing during which Association of Public Health Labora- ease, adequate testing and regulato-
contamination is likely controlled? tories put out an excellent guide out- ry limits can be the literal difference
Although these questions seem lining potential contaminates in vari- between life and death (6,11,12).
like the logical place to start, regula- ous cannabis product (see Table I) (2). Regulators also should take into
tors often are challenged with getting Regulators and stakeholders must consideration product type with re-
rules into place quickly, typically only take into consideration what the food lation to the target analyte being
months before a product is allowed and environmental industries have mandated. Although it might not be
to be offered for sale. Thus, rules may required for similar items, which are necessary to test bud intended for
be drafted and implemented based generally based on product type (pro- smoking for Staphylococcus aureus,
on worst case scenario only. In fact, cess type) as well as the end product it is definitely warranted to test for it
draft regulations have even listed user. For regulators, it is extremely in products intended for vaginal or

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 Featture 25

cutaneous use because it can cause guidance and outlines various sam- ~90% confidence, and 15 subsam-
skin and soft tissue infections (2). In ple amounts taken for Salmonella de- ples for ~80% (16,17). Sample sizes are
conjunction with product intended pending on food type and intended combined to form an analytical unit
use, it is important to consider out- consumer (6). In compendial micro- (generally 25 g or more). However, in
breaks that have occurred in other bial sampling protocols, the required cannabis, samples often are too few,
industries. For some reason or an- sampling amount includes at least 60 not random, and too small. For exam-
other, several organisms have rare- subsamples to achieve ~95% confi- ple, eight subsamples combined to
ly been included as cannabis regula- dence in a batch, 30 subsamples for form a composite test sample (even if
tory testing requirements, but should
be evaluated in risk assessment. Two
prime examples are Listeria monocy-
togenes and Mucor spp (6,11,). Prop-
er testing must include the appropri-
DON’T LET QUALITY
ate mandated targets and testing for
microbial targets must be mandated
GO TO THE DOGS
for the correct reasons. A great ex-
ample of an incorrect assumption is
not testing for Salmonella simply be-
cause a product’s water activity is
low; low water activity does guaran-
tee stability and bacterial stasis, but
it never demonstrates a lack of via-
ble organism. This phenomenon was
clearly demonstrated with the 1993
Salmonella outbreak in Germany that
occurred in potato chips. More than
1000 people were reportedly affect-
ed by Salmonella determined to have
originated from dried paprika pow-
der, which after eight months of dry
storage still contained 0.7 CFU/g of
Salmonella (9).
The other major challenge in the FEED YOUR EQUIPMENT H2, N2
AND ZERO AIR ON-DEMAND
area of proper regulation is testing
frequency. Regulatory agencies have,
in general, struggled with the amount
of cannabis material to require for • Consistent Purity
submission to laboratories—and at • Consistent Pressure
what frequency—because of the
cost of the product. Although there
• Proven Safe
are other industries from which to • Cost Effective
draw, as it stands in cannabis, most • Eliminates Cylinder Storage
sampling sizes (where often a single
gram is submitted for microbial test-
and Delivery Issues
ing) cannot be considered statistical-
ly relevant (16,17). Sampling statistics, Visit us on-line or call for a consult
regardless of the level of tolerance, with one of our sales engineers today.
must be based on the likelihood of
occurrence as well as the risk to the
end consumer. The U.S. Food and
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Drug Administration (FDA) offers www.ProtonOnSite.com

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

26 Feature

especially in products in which the or-

Actin Actin/nuclei Cell wall ganisms are stressed. One study by
(a) (b) (c) Beuchat and colleagues (8) found a
difference in performance between
3M Rapid Pertifilms when compared
WT with 3M traditional seven-day films
and Dichloran Rose bengal chloram-
phenicol (DRBC). For certain matri-
ces, 48-h counts could be several log-
(d) (e) (f)
arithms different than those same
matrices taken to 72 h or 120 h. For
Scact1
example, the authors found dried ba-
nana chips produced <1 log-count
yeast at 48 h and 72 h, but around
6-log count of yeast at 120 h (8). Al-
though 48-h counts might work well
in some cannabis products, just as
48 h has been shown acceptable in
Figure 1: Differential support of Aspergillus fumigatus morphogenesis by yeast and hu-
certain food products, it is probable
man actins.
that such short timeframes may not
work in every cannabis matrix (13,19).
equaling 25 g) would provide a mere important for laboratories to recog- More modern technology, such
60% confidence that the batch was nize the limitations of microbial test- as molecular DNA-based methods,
not contaminated; or, considered ing and follow well established con- offer even faster turnaround times,
another way, with only eight sample ventions, because these conventions but the conversion between genom-
units taken, at least 40% of the en- are not exclusive to a single disci- ic copies or units (GU) and colony
tire batch would need to be contam- pline. Turnaround times are probably forming units must be further estab-
inated with target in order for a pos- the larger of the two issues. As clients lished and shown as statistically com-
itive laboratory result to occur (16). It push for results as quickly as possi- parable. Often, these methods tend
is strongly recommended that reg- ble, it is tempting to jump on the fast- to overcount or overestimate certain
ulators work with experts in the field est method that may or may not be molds, possibly depending on the
of microbial sampling and risk analy- able to produce results as accurately number of nuclei found within a sin-
sis to determine the best approach, as slower compendial methods. With gle CFU. This problem becomes ap-
ensuring that sample sizes and test- regard to fast laboratory turnaround parent when presented with molec-
ing have significance. Sample sizes of times, yeast and mold testing is gen- ular data that clearly and accurately
<10 sample locations providing only a erally the bottleneck, and, as a result, counts yeast concentrations, but
small laboratory sample are not rec- several new methods have come to shows mold CFU counts as double
ommended and, in all probability, are the forefront in response to the can- (or more) when compared with agar
meaningless (especially when taking nabis industry’s call for faster testing. based methods. This nuclei problem,
into account laboratory subsampling). The most common yeast and mold in all likelihood, could be overcome,
method used by far are the rapid but only with very careful conversion
Common yeast and mold plates, such as those calculations. To illustrate this, Figure
Methodological Challenges produced by 3M and IDEXX (Sim- 1 shows Aspergillus fumigatus nu-
The cannabis industry also faces Plate). It is important, when consid- clei (small red spots identified by the
methodological challenges. Turna- ering these methods, to validate small white arrow in Figure 1e) con-
round times are constantly pushing against seven-day methods in natu- tained within a single CFU (10).
test methods to the brink of time lim- rally contaminated product to estab- Molecular methods also have the
its. Further, microbiology norms are lish that similar results occur regard- added challenge of pulling DNA both
often ignored as the cannabis indus- less of timeframe. It has been shown from only living tissue and spores
try sometimes treats microbial test- that faster methods don’t always per- while simultaneously excluding dead
ing as a completely new field. It is form the same as slower methods, DNA that would not show up on agar

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 Featture 27

based methods. Although there is Molecular methods possess their are a true and legitimate problem.
nothing inherently wrong with any of own established norms and should At the same time, it is always recom-
these methods in theory, it is up to include important control factors mended to choose a method that has
laboratories, regulators, and vendors such as internal amplification con- some basis in other regulatory fields,
to ensure the methods produce sim- trols (manufactured in such a man- such as food or environmental, rather
ilar results as compared with more ner and provided at a low concentra- than to develop a completely new in-
established counterparts (culture- tion so as not to compete with target house method from scratch with limit-
based methodologies). DNA). Newer molecular methods also ed validation funding. It is common in
As mentioned earlier, another should be rigorously tested against the cannabis industry to attempt the
common challenge facing the testing an array of organisms and matrices to latter; the thinking being that, as can-
of cannabis is a dismissal of microbial ensure that cross reactivity and false nabis is a new industry, so too must all
conventions—microbiology has cer- negative are acceptably low (1,5,7). the methodology be new and propri-
tain statistical probabilities and test- These challenges are, however, eas- etary. But an AOAC standard meth-
ing conventions that do not match ily rectified because conventions ex- od that includes 100 serovars of Sal-
those found in chemistry. It is not ap- ist and are readily available in other monella (50 for other targets) and 50
propriate in microbiology to report industries. It is highly recommend- strains of exclusivity bacteria is always
values with more than two signifi- ed that laboratories adopt microbial preferred from a regulatory stand-
cant figures because certainty dras- conventions as published by the U.S. point when compared to an in-house
tically decreases past two significant Department of Agriculture (USDA), method that was tested against what
figures. For example, a plate count FDA, American Herbal Products As- the laboratory could afford (often
might indicate 111,111 CFU/g, but the sociation (AHPA), AOAC, and oth- less than 10 strains of target and few-
actual “true” value, should the sam- er sources, especially regarding the er than 10 strains of nontarget) (1).
ple be plated to infinity, would be number of inclusivity and exclusivity This, of course, is not to discourage
closer to 110,000 (4,18). In addition, organisms required to ensure meth- the development of new methodol-
it is important to recognize limits of ods are as robust and well-rounded ogy, but care must be taken to en-
quantitation and detection. It is nev- as possible (1,5). sure method parameters and controls
er appropriate to count plate grids or meet those of highly robust and es-
estimate counts and report this as the Matrix Challenges tablished methods that might only re-
actual counted value. Counts such as Cannabis is an unequally challenging quire a matrix extension.
this require qualifiers such as “great- matrix and, while the closest relative Regardless of the method’s origin,
er than” or “estimated” (14,16,18). It is probably food, even food is not en- it is extremely important for laborato-
also is considered convention to an- tirely comparable in terms of product ries to test methodology against as
alyze quality controls using logarith- complexity. Cannabis matrices arrive many matrices as possible to weed
mic converted values to account for at the laboratory in a vast and end- out potential problems. The use of
the natural uncertainty encountered less number of variations. As such, it surfactants is advised for methods
when working with living systems (16). is immensely important to validate designed for the analysis of highly
Further, it is convention in microbiol- methods to include as many matri- fatty “budders,” waxes, or oils (4). It is
ogy to only retest a sample because ces as possible, especially for those important to remember that inhibito-
of quality control failures—it is nev- that laboratories expect to encounter ry agents are a common occurrence
er acceptable to retest client samples frequently. Simply assuming a meth- in microbial testing and it is neces-
simply because the client did not like od that works in marijuana bud and sary to dilute out potential inhibitory
the result. Retests will often produce brownies will work in “budder” con- agents. This approach is well known
different results because of storage centrates is not reasonable (5). It is in food and other industries in which,
time and conditions. If a retest is per- advised to follow AOAC, USDA, and for example, items such as oregano
formed, acceptance criteria must be FDA validation guidelines for micro- and cinnamon must be well diluted
established as to how the retest re- biology as closely as possibly within to ensure recovery of the target (iron-
sults should compare with the origi- budget. It may not be possible for a ically, the inhibitory nature of a prod-
nal result. A change from a result of laboratory to validate a method to the uct does not always protect the con-
too numerous to count (TNTC) to a level of an AOAC compendial meth- sumer from contaminate bacteria)
retest value of <1 CFU/g is never ac- od on a startup budget. Regulators (4,12). The importance of matrix ef-
ceptable (14). should remember financial limitations fects must be taken into account. It

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28 Feature

standard/45035.html (ISO, 2011).

should never be assumed one matrix References
represents many or even a few. 8) L.R. Beuchat and D.A. Mann, J.
1) Association of Analytical Food Prot. 79(1) 95–111 (2016).
Communities (AOAC), “Methods
Conclusion Committee Guidelines for Validation 9) L.R. Beuchat, E. Komitopoulou, H.
Microbial testing in the cannabis in- of Microbiological Methods for Food Beckers, R.P. Betts, F. Bourdichon,
and Environmental Surfaces,” http:// S. Fanning, H.M. Joosten,
dustry faces many hurdles, most of www.aoac.org/imis15_prod/AOAC_ and B.H. Ter Kuile, J. Food
which are simply growing pains that Docs/StandardsDevelopment/ Prot. 76(1), 150–172 (2013).
will be worked out over time as the AOAC_ Validation_Guidelines_
for_Food_Microbiology- 10) L.L. LeClaire and J.R. Fortwendel,
industry gains acceptance and con- PLoS One 10, 11 (2015).
Prepub_version.pdf (2012).
ventions are established. Of the chal-
2) Association of Public Health 11) M. McCarthy, A. Rosengart,
lenges outlined, sampling statistics is A.N. Shuetz, D.P. Kontoyiannis,
Laboratories (APHL), “Guidance
arguably the most important, but, as and T.J. Walsh, N. Engl. J.
for State Medical Cannabis Testing
more and more individuals with ex- Programs,” https://www.aphl. Med. 371, 150–160 (2014).
pertise in the area enter the indus- org/aboutAPHL/publications/ 12) S. Mortimore and C. Wallace,
try, microbial testing will likely be Documents/EH-Guide-State-Med- HACCP: A Practical Approach,
Cannabis-052016.pdf (2016). 3rd Ed. (Springer, Springer
resolved. With regard to sampling,
3) “Colorado Retail Marijuana Code,” New York, 2013).
regulators should seek experts—and
stand against the industry when nec- Adpoted Rules, CCR 1 212-2, Rule 13) P. Bird, J. Flannery, E. Crowley,
712, Section R 712 – Retail Marijuana J. Agin, and D. Goins,J. AOAC
essary—to prevent a potentially un- Testing Facilities: Sampling and Int. 98(3), 1563–1575 (2015).
warranted push to get sample sizes Testig Program, E. Permissable
as low as possible, often with no con- Levels of Contaminats (Adpoted 14) R.A. Cowie, Quality Assurance
November 17, 2017; Effective for Microbiology in Feed
sideration of the threshold at which
January 1, 2018), pp. 124–125. Analysis Laboratories, (Food and
samples no longer have meaning. As Agriculture Organization of the
for cannabis testing methodology, 4) US Food and Drug Administration, United Nations, Rome, 2013).
(FDA), Bacteriological Analytical
methods must be well vetted and, if 15) Rules and Regulations of the State
Manual (BAM), https://www.fda.
developed internally, derived from gov/Food/FoodScienceResearch/ of New York, § 55-2.15 Requirements
an applicable method available from LaboratoryMethods/ucm2006949. for laboratories performing testing
other fields of testing. As laborato- htm (FDA, Rockville, Maryland, 2016). for medical marihuana (Regulatory
Amendments, Effective December
ry regulation continues to increase 5) US Food and Drug Administration, 27, 2017), 1004.13 (g) 32-33.
and conventions are reached, micro- Office of Foods and Veterinary
Medicine, Guidelines for the 16) Y. Salfinger and M.L. Tortorello,
bial testing challenges will be fewer
Validation of Analytical Methods Compendium of Methods for
and fewer. However, until that point for the Detection of Microbial the Microbiological Examination
is reached, the majority of challeng- Pathogens in Foods and Feeds of Foods, 5th Edition (American
es are faced by cannabis laboratories 2nd Edition, http://www.fda.gov/ Public Health Association, 2015).
that are directly impacted by fast reg- downloads/ScienceResearch/
17) S. Dahms, ”Microbiological
FieldScience/UCM298730.pdf
ulatory changes and are under pres- Sampling Plans – Statistical Aspects”
(FDA, Rockville, Maryland, 2015).
sure to keep clients by offering fast presented at the 36th Symposium of
6) US Food and Drug Administration, the Swiss Society of Food Hygiene,
turnaround times. Thus, it is impor- Zurich, Germany (2003), pp. 33–36.
Office of Regulatory Affairs:
tant for regulators to keep in mind Inspections, Compliance,
18) S. Sutton, Journal of Validation
the impact of rapid testing chang- Enforcement, and Criminal
Technologies 17(3), 42–46 (2011).
es, while at the same time ensuring Investigations, Investigations
Operations Manual, Chapter 4 - 19) V.H. Tournas, L. Feliciano,
that regulations have meaning. It is
Sampling, https://www.fda.gov/ and E.J. Katsoudas, J. Food
in the best interest of both stake- downloads/ICECI/Inspections/ Saf. 30, 506–514 (2010).
holders and regulators to work to- IOM/UCM123507.pdf (FDA,
gether to ensure testing regulations Rockville, Maryland, 2018).
Matthew A. Ward
are meaningful (statistically, logical- 7) International Standards Organization is the Marijuana Laboratory Auditor
ly, and methodologically), and there- ISO 22119 Microbiology of food with the Colorado Laboratory Services
fore stable, while keeping in mind and animal feed stuff – Real-time Division, Department of Public Health
polymerase chain reaction (PCR) & Environment in Denver, Colorado.
cost, as well as ensuring consumers for the detection of food borne Direct correspondence to:
receive quality product that is safe for pathogens – General requirements [email protected]
consumption. and definitions, https://www.iso.org/

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

30 Feature

Selecting Microwave Digestion

Technology for Measuring Heavy
Metals in Cannabis Products
Microwave digestion is an excellent tool that provides complete dissolution for the accurate and
precise testing of heavy metals in cannabis and its products using plasma spectrochemical techniques,
such as inductively coupled plasma–optical emission spectrometry (ICP-OES) and inductively coupled
plasma–mass spectrometry (ICP-MS). However, there are many different options of commercially
available microwave digestion systems, each with their own strengths and weaknesses. The optimum
choice will often depend on the workload and sample diversity of the cannabis testing laboratory
carrying out the analysis. This article describes the basic principles of the major types of microwave
digestion technology and offers suggestions as to which might be the best approach based on sample
matrix, digestion efficiency, sample throughput, productivity, and overall cost of analysis. The article is
supplemented by real-world examples of how different microwave technologies are being utilized in
cannabis testing laboratories for the digestion and measurement of heavy metals by ICP-MS.

Ryan Boyle and Eric Farrell

W
ith increased state regu- reaction chamber (SRC) technology potential matrix-suppression effects
lations, cannabis growers (1). So, how do you go about select- in the plasma should be taken into
are required to conduct ing the optimum technology for your consideration. It is therefore well-rec-
trace metals testing to ensure a safe samples? What types of mineral ac- ognized that the most plasma spec-
and high-quality product. This test- ids will be best suited for your ele- trochemical-friendly reagents are
ing encompasses a wide variety of ments of interest, and what temper- typically strong oxidizing agents
samples from growers and the pro- ature and pressure will be required such as nitric acid (HNO3) and hydro-
cessing industry including soils, fer- for the digestion process of your gen peroxide (H2O2), which are ex-
tilizers, plant material, edible prod- sample matrices? It’s only when you tremely efficient, but tend to gener-
ucts, concentrates, and topicals. have a good understanding of these ate large amounts of carbon dioxide
Obtaining analytical data required issues, that you can begin to look (CO2) and various oxides of nitrogen
to ensure quality products starts more closely at the pros and cons of (NOx) when they react with the sam-
with the crucial step of preparing the different commercially-available ples. The microwave system and its
the sample for analysis. Reducing microwave technology. So first, let’s components will therefore not only
handling steps, eliminating outside take a closer look at the fundamen- need to accommodate the high tem-
contamination, and minimizing rea- tal principles of both designs. perature required to digest all the dif-
gent blank contribution are all nec- ferent organic sample types, but also
essary for good sample preparation. Principles of Microwave be able to handle the subsequent in-
It is well recognized that closed-ves- Digestion Technology crease in pressure produced by the
sel microwave digestion offers the First it should be emphasized that generation of large volumes of these
best approach for getting your sam- sample digestion of organic matrices gases. For some samples, the addi-
ples into solution for analysis by in- such as cannabis products should be tion of small amounts of hydrochloric
ductively coupled plasma–optical carried out using reagents compati- acid will also help to stabilize some el-
emission spectrometry (ICP-OES) or ble with ICP-OES and ICP-MS instru- ements particularly mercury (Hg) and
inductively coupled plasma–mass mentation. For example, the chemical the platinum group elements. How-
spectrometry (ICP-MS). However, or physical properties and concentra- ever, it should be noted that if ICP-MS
there are basically two very differ- tion of the mineral acids used and is being used as the analytical tech-
ent commercially available designs: how they affect the sample introduc- nique the 40Ar35Cl polyatomic spe-
rotor-based systems and single tion nebulization processes and the cies could potentially interfere with

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 Featture 31

monoisotopic arsenic (As) at 75 atom-

ic mass units (amu). This interference
can be alleviated using a collision–re- (a) (b) (c)

action cell (CRC), but it is important to

be aware of this potential problem so
that the optimum instrumental condi-
tions are used.

Rotor-Based Technology
With rotor-based technology, micro-
waves are directed onto vessels con-
taining the sample and the digestion
reagents, which are placed in a rotat-
ing carousel. The digestion process
is accomplished by raising the pres-
sure and temperature through micro- Figure 1: Principles of the “vent-and-reseal” approach.
wave irradiation, as the carousel is ro-
tating. This increase in temperature
and pressure, together with the op- 40
timum reagent, increases both the
speed of thermal decomposition of 30
Pressure (bar)

the sample and the solubility of met-

als in solution. Rotor-based systems 20
work extremely well for similar matri-
ces by batching all samples togeth- 10
er that react in the same way. By car-
rying out the digestion process using 0
the same microwave power, temper- 0.00 0.01 0.02 0.03 0.04
ature, and pressure conditions, it will Time (h.mm)
ensure similar digestion quality in Vent and reseal Burst disk
all positions. To increase through-
put, different sized carousels can
be used depending on the sample Figure 2: Comparison of the burst disk approach with “vent-and-reseal.”
workload. However, when many dif-
ferent sample matrices have to be di- • Self-regulating (Figure 1c), and the digestion contin-
gested, productivity could be sacri- Let’s take a closer look at how they ues. There is no loss of sample and no
ficed, because each sample type has work. clean up needed. The microwave pro-
to be batched together, which unfor- gram continues to completion and no
tunately precludes different samples Vent-and-Reseal rerun is required.
being digested together in the same This patented technology (3) eliminates
sample run. vessel failure in the case of an out of Burst Disk
Additionally, the strength of any control exothermic reaction. Figure 1 This method uses a simple burst disk
rotor-based system is the ability to shows “vent-and-reseal” in action: the in the cap that is designed to fail in
achieve high pressure and safely vent vessel cap is held in place by a dome- an over-pressure situation, instant-
the excess pressure caused by the shaped spring (Figure 1a). In the case of ly releasing all pressure in the vessel.
buildup of CO2 and NOx during the over pressurization because of a high- When this happens, instantaneous
digestion process. There are basical- ly exothermic reaction, the spring is flat- boiling occurs, the sample contents
ly three different approaches used to tened, allowing the cap to lift up slight- are lost, and the run has to be man-
carry out this process (2). They are: ly (Figure 1b), releasing excess pressure. ually stopped. The result is that a sig-
• Vent and reseal Immediately, the excess pressure is re- nificant cleanup of the cavity is re-
• Burst disk leased, the spring reseals the vessel quired, with a strong possibility that

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

32 Feature

environmental, and food. Most re-

Pressure release
N2 in (at end of run) cently, the self-regulating approach
has been applied to high pressure
rotors designed specifically for more
Clamp
challenging sample types. Since self-
regulating vessels are designed to
vent, incomplete digestions may be
Samples sit in liquid - constant “load” for
PTFE
Sample microwave from run to run observed given that the pressure loss
Load does not allow the required temper-
Isolation
Water cooling atures necessary for complete diges-
Magnetron
tion to be achieved.

Microwave
Single Reaction
Chamber Technology
So let’s take a more detailed look
Figure 3: Single reaction chamber microwave digestion technology. at the principles of single reaction
chamber (SRC) technology, and how
it differs from the rotor-based sys-
tem. Instead of a rotor with a dis-
crete sample vessel, the samples are
Hg (ppt)
put into vials with loose-fitting caps,
Bk position 01 0.020
which are sitting in a rack that is low-
Bk position 03 0.032
ered into a larger vessel containing a
Bk position 05 0.001
base load of acidified water. It’s this
Bk position 07 0.002
baseload that absorbs the micro-
Bk position 09 -0.003
wave energy and transfers it to the
Bk position 11 -0.007
vial. This design allows every vial to
Bk position 13 -0.007
(15 Position) White vials: blanks. react independently within the base
Red vials: 110 ppm Hg Bk position 15 -0.006
load and ensures that all samples
achieve maximum temperature with
pressures contained up to 200 bar.
No batching of samples is neces-
Figure 4: Study of sealing capability of nitrogen gas shows no sign of contamination sary, and any combination of sample
from mercury. type and acid chemistry can be run
simultaneously in the same chamber.
corrosion of internal components will in high-throughput rotors, which were A schematic of the SRC is shown in
occur. This is exemplified in Figure 2, developed to address the needs of Figure 3.
which compares the burst disk ap- laboratories that process larger sam- As previously mentioned, loose-
proach with vent-and-reseal: the red ple volumes on a routine basis. Self- fitting caps are used to seal the vi-
line shows the complete loss of pres- regulating vessels are very easy to as- als. This is possible because they are
sure when a burst disk fails, and the semble and disassemble and rely on pre-pressurized with 40 bar of nitro-
whole run is stopped. Whereas, the the PTFE sealing plug inside the cap gen before the start of the micro-
blue line shows that with the vent- deforming to release pressure. Their wave program, which acts as a gas
and-reseal technology, excess pres- compact design results in more mod- cap and keeps all the vials indepen-
sure is gently released at the vessel’s erate temperature and pressure ca- dently closed. As the pressure builds,
pressure limit (f.i. 30 bar), and the di- pabilities, but allows for a large num- equilibrium is achieved both inside
gestion continues to completion, with ber of vessels to fit onto the rotor. and outside the vial. As a result, a va-
no loss of sample. These characteristics make self-reg- riety of vial types including dispos-
ulating rotors ideal for high workload able glass, quartz, and PTFE or any
Self-Regulating Rotors laboratories with relatively straight combination of these materials can
This third approach is typically used forward applications such as clinical, be used (4).

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 33

Benefit of Nitrogen Table I: Range of cannabis products tested, their sample weights, and digestion
Pressurized Caps reagents using the rotor-based microwave system
To exemplify that 40 bar of nitrogen Sample Weight Used in Ethos UP – SK15
is sufficient to seal the vials, an ex- Sample
8 mL Conc. HNO3, 1 mL Conc. HCl, 1 mL H2O2
periment was carried out where vials Cannabis plant material (flowers) 1g
containing 110 ppm of mercury were
CBD oil (cannabidiol) 0.5 g
placed right next to blank solutions
Cannabis vape cartridge 0.5 g
in a 15-rack sample holder. In other
words, every alternate sample vial in Cannabis salve (ointment) 0.5 g

the rack contained either 110 ppm or Cannabis flavored cookies 1g

0 ppm mercury. It is well recognized Cannabis flavored gummy bear 1g
that mercury is highly volatile, particu-
larly when heated, and would contam-
Table II: Rotor-based microwave program used to digest samples
inate any surrounding vessels if not
Step Time (min) Temp. Power
capped tightly. However, it can be
seen in Figure 4 that the measurement 1 20:00 210 °C 1800 W
of every alternate blank sample by 2 15:00 210 °C 1800 W
ICP-MS is actually at the limit of detec-
tion for the technique. This is a clear Major Differences Between requirements are different. So be-
indication that SRC technology using Rotor and SRC Technology fore we focus on some real-world
pressurized nitrogen gas as a sealant It’s important to emphasize that eve- applications of cannabis testing
eliminates the potential of cross con- ry laboratory’s trace element analy- for heavy metals, let’s remind our-
tamination in the sample chamber. sis workload and sample digestion selves of the differences between
34 Feature

Table III: Recovery of 20 ppb Pb, As, Cd, and 10 ppb Hg spiked into cannabis products, digested by rotor-based microwave technology,
and analyzed by ICP-MS

Cannabis Product Analyte As Cd Hg Pb

Cannabis % Recovery 91.8 92.3 101.4 98.7

plant material % RSD (n = 3) 2.3 0.7 1.3 1.5
% Recovery 91.3 87.3 105.8 97.3
CBD oil
% RSD (n = 3) 2.7 2.8 1.4 2.3

Cannabis % Recovery 94.5 92.8 99.3 102.5

vape cartridge % RSD (n = 3) 1.5 2.2 1.2 1.5
% Recovery 90.7 95.8 102.3 89.2
Cannabis salve
% RSD (n = 3) 2.1 1.0 1.5 2.5

Cannabis % Recovery 92.2 96.2 95.6 93.5

flavored cookies % RSD (n = 3) 2.1 1.3 1.4 1.0

Cannabis flavored % Recovery 97.8 96.7 98.2 96.7

gummy bear % RSD (n = 3) 2.0 0.3 0.4 1.8

rotor-based systems and SRC tech- normal plant metabolism. Some of metals. Depending on their work-
nology. The main drawback of ro- these metals are naturally occurring load and the diversity of the products
tor-based technology is that they and leach into the groundwater. Oth- and testing protocols used will clear-
require batching of similar matrices ers precipitate in rainwater or may ly impact the optimum type of mi-
and chemistries, because control of be introduced into the plant’s envi- crowave system they choose for this
the power is based on the reaction ronment as constituents of fertiliz- task. To get a better understanding
of one vessel at a time. Regardless ers, pesticides, herbicides, and fun- of this, we took a representative sam-
of how temperature is being meas- gicides used to increase crop yield. pling of different commercially availa-
ured, it’s still measuring the pow- Regardless of their prevalence, when ble cannabis products and digested
er level based on one sample. By metabolized, metals are absorbed them by both rotor-based technolo-
batching similar samples, under-di- and transported through the plant gy (Ethos UP- SK-15 high pressure sys-
gestion of some samples can be roots into plant tissue, leaves, and tem, Milestone Inc.) and a single re-
minimized because of the pressure flowers. Cannabis is so effective at action chamber system (UltraWAVE,
and temperature required by oth- absorbing metals from its environ- Milestone Inc.) and analyzed them
ers. Additionally, in rotor-based sys- ment that it has been referred to as using ICP-MS (Model 7900, Agilent
tems, the vessels are typically made a hyper-accumulator of trace met- Technologies).
from PTFE, which limits the temper- als, including many of the tradition-
ature and pressure because of the al heavy metals. This characteristic of Rotor-Based Analytical Procedure
type of material used. The multiple cannabis leads to concern that these Table I shows the cannabis products
components of vessels also require elements may occur in high concen- tested, their sample weights, and re-
additional handling before and after trations in cannabis plants. State gov- agents used to digest them using
digestion, which could impact pro- ernments and private laboratories are the rotor-based microwave system,
ductivity. Depending on detection therefore focusing on product safety and Table II exemplifies the micro-
limit requirements, the vessel liners testing with special emphasis on As, wave program that was used to di-
may also require extra cleaning be- Cd, Hg, and Pb because they are ex- gest the samples. The resulting solu-
tween runs. tremely hazardous to human health, tions produced were then made up to
even at low levels. 50 mL with deionized water and pre-
Heavy Metals Testing of The combination of closed-vessel sented to the ICP-MS instrument for
Cannabis Plants, Concentrates, microwave digestion and ICP-MS has analysis. Note: only Pb, Cd, As, and
and Edibles by ICP-MS become the preferred approach of Hg will be presented here, but a to-
Like all plants, cannabis absorbs met- cannabis testing laboratories to carry tal of 14 elements (As, Cd, Pb, Hg, Ag,
als from its environment, a result of out the measurement of these heavy Ba, Co, Cr, Cu, Mn, Ni, Se, V, and Zn)

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 35

were evaluated. Please refer to the fol-

Table IV: Range of cannabis products tested, their sample weights, and digestion
lowing reference for more information reagents using the SRC microwave system
about the methodology and the full
Sample Weights Used in the UltraWAVE
data set (5). Sample
4 mL Conc. HNO3, 1 mL Conc. HCl
Cannabis plant material (flowers) 1g
Results and Discussion
CBD oil (cannabidiol) 1g
The performance of the rotor-based
system was evaluated through a recov- Cannabis vape cartridge 1g

ery study on samples of interest for the Cannabis salve (ointment) 1g

cannabis industry, from plant material Cannabis flavored cookies 1g
to edibles and concentrates. All sam- Cannabis flavored gummy bear 1g
ples were fortified with a spike solu-
tion containing 20 ppb of all analytes,
except for Hg, which was 10 ppb. The Table V: SRC microwave program used to digest samples
analytical results are shown in Table
Step Time (min) Temp. 1 Temp. 2 Pressure Power
III with recoveries of all elements be-
1 20:00 240 °C 60 °C 110 bar 1500 W
tween 87–106% and relative standard
deviations (RSDs) well below 3%. 2 10:00 240 °C 60 °C 110 bar 1500 W

Simultaneous Mixed-Batch simultaneously without sample-to- point out that higher sample weights
Sample Preparation for Metals sample cross contamination. Addi- are typically used with SRC technol-
Testing of Cannabis Samples tionally, a soil sample was digested ogy and there is no minimum sample
Using SRC Technology in the same analytical run as the can- volume that has to be used. In addi-
The combination of SRC technolo- nabis products, to exemplify the ca- tion, because of the higher temper-
gy with ICP-MS allows cannabis an- pability of SRC technology to digest ature and pressure capability of the
alytical testing laboratories to an- widely different matrices simultane- SRC technology, digestion times are
alyze a broad variety of sample ously in the same chamber. This ap- usually much shorter.
matrices with widely different sam- proach would be very difficult to car-
ple weights and elemental require- ry out with a rotor-based system, Results and Discussion
ments. SRC microwave digestion is because of its batching limitations. The performance of the SRC system
a unique approach, incorporating all was evaluated through a recovery
the benefits of closed-vessel micro- Analytical Procedure study on samples of interest for the
wave digestion while making sample Using SRC Technology cannabis industry, from plant ma-
preparation fast, easy, and extreme- Table IV shows the cannabis prod- terial to edibles and concentrates.
ly efficient. SRC technology operates ucts tested, their sample weights All samples were fortified with a
at very high temperatures and pres- and reagents used to digest them spike solution containing 20 ppb of
sures (up to 300 °C and 200 bar, re- using SRC technology, and Table V all analytes, except for Hg, which
spectively), allowing for complete di- exemplifies the microwave program was 10 ppb. A soil reference mate-
gestion of even large sample sizes that was used to digest the sam- rial (SRM 2711a) was also included in
(up to 3–5 g) and difficult-to-digest ples. The resulting solutions pro- this study as a quality control sam-
matrices. Samples can be weighed duced were then made up to 50 mL ple and to show that a widely dif-
directly into disposable glass vials, with deionized water and presented ferent matrix to cannabis products
eliminating the need for acid clean- to the ICP-MS instrument for analy- could be digested in the same sam-
ing and vessel assembly. Further- sis. Note: As with the previous meth- ple run. It should be noted that the
more, SRC eliminates the need to od, only Pb, Cd, As, and Hg will be recoveries in the soil were calculat-
batch samples because mixed sam- shown here, but 14 elements (As, Cd, ed according to the total element
ple types, weights, and acid chemis- Pb, Hg, Ag, Ba, Co, Cr, Cu, Mn, Ni, content and represent the leacha-
tries can be successfully processed Se, V, and Zn) were evaluated. Please ble fraction, based on the acid used
in the same run. This application de- refer to the following reference for . . . refer to The National Institute of
scribes how a variety of samples from more information about the method- Standards and Technology (NIST)
the cannabis industry were digested ology used (6). It’s also important to Certificate of Analysis for SRM 2711a

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

36 Feature

Table VI: Recovery of 20 ppb Pb, As, Cd, and 10 ppb Hg spiked into cannabis products, digested by SRC technology, and analyzed by
ICP-MS

Cannabis Product Analyte As Cd Hg Pb

Cannabis % Recovery 91.7 93.0 98.7 88.3

plant material % RSD (n = 3) 1.9 2.1 2.1 2.6
% Recovery 95.8 98.5 97.6 89.7
CBD oil
% RSD (n = 3) 1.8 2.3 1.1 2.2

Cannabis % Recovery 90.8 87.3 91.8 92.0

vape cartridge % RSD (n = 3) 1.1 2.0 1.2 1.5
% Recovery 95.8 91.5 94.3 95.3
Cannabis salve
% RSD (n = 3) 0.3 1.1 1.4 2.2

Cannabis % Recovery 92.8 93.8 96.1 93.3

flavored cookies % RSD (n = 3) 2.8 0.7 1.3 1.4

Cannabis flavored % Recovery 90.2 89.5 94.1 91.8

gummy bear % RSD (n = 3) 2.1 2.0 1.0 2.2
Leachable
89 47 7.4 1300
Conc. (mg/kg)
Soil (SRM 2711a)
% Recovery 90.4* 94.1* 98.7* 93.3*
% RSD (n = 3) 2.1 1.9 1.6 1.1
* Note recovery based on leachable certificate values.

for further details (7). The analyti- found in cannabis plants and relat- if there is a need to digest many dif-
cal results for the cannabis prod- ed products. Highly reactive sam- fering sample matrices simultane-
ucts and soil reference sample are ples such as gummy bears, cookies, ously in the same run, an SRC system
shown in Table VI with recoveries of and cannabidiol (CBD) oil have been could be the best solution. Because
all analytes between 87.5–98.7% and completely digested even in large of its higher sample capacity, use
RSDs well below 3%, clearly demon- sample amounts, ensuring reliable of disposable vials, and faster cool
strating the robustness and repro- analysis. In addition to good ana- down times, sample throughput pro-
ducibility of microwave digestion lyte spike recoveries, this approach, cessing with SRC technology is up to
of different sample types using SRC using a high pressure, rotor-based 2–3 times higher than convention-
technology. Note: The full data set system with a 15 position carousel al closed-vessel digestion systems.
for all 14 analytes can be viewed at also provides a high level of repro- The ability to digest different sam-
the following reference (6). ducibility, even for volatile elements ple types together and larger sam-
such as As and Hg. And if higher ple weights with minimal acid vol-
Final Thoughts sample throughput is required for ume makes it the optimal technique
Modern plasma spectrochemical less demanding matrices, it should to perform sample preparation for
techniques will generate trace el- be noted that a 44-position option all cannabis related products, from
ement data of the highest quali- is available. the plant material to edibles, con-
ty. However, with any solution tech- However, for contract laboratories centrates, and even to get a better
nique, the data will only be as good that might be handling a more di- understanding of the soil chemistry
as the sample presented to the in- verse and complex range of samples where the plants are growing.
strument. For that reason, robust such as supplements, pharmaceu- There is no question that a new era
closed-vessel microwave diges- tical, or even soil samples, a rotor- of acceptance and legalization has
tion techniques are critically impor- based system might be somewhat opened new opportunities for can-
tant to the overall analytical proce- restrictive, because similar samples nabis testing laboratories, not only in
dure. The data in the first part of have to be batched together to en- heavy metals but also for an expand-
this study demonstrates full recov- sure they are being digested under ed list of analytes including poten-
ery of the most common elements the optimum conditions. Therefore, cy, terpenoids, pesticides, residual

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 37

2) “Microwave Digestion Vessel 7) The National Institute of Standards

solvents, moisture, mycotoxins, and Venting Technology,” Milestone and Technology (NIST) Certificate
pathogens. Standardization of these Inc., https://milestonesci.com/ of Analysis for SRM 2711a (Montana
methods for the industry will give ethos-up-venting-technology/. Soil): https://www-s.nist.gov/
regulators the resources and infor- srmors/view_cert.cfm?srm=2711A.
3) W. Lautenschlager, Milestone Srl,
mation they need to include com- U.S. patent 5,270,010, 1990-06-13. 8) “Milestone’s UltraWAVE allows
mon sense regulations and legisla- Cannabis Lab to Streamline Heavy
4) “Any Sample Any Time: The Metals Testing: CannaSafe Labs,
tion to monitor and control the use Benefits of Single Reaction Case Study,” https://milestonesci.
of cannabis within the United States’ Chamber Microwave Digestion for com/cannabis-testing/.
medicinal and recreational markets. Trace Element Analysis: A White
Paper,” Milestone Inc., https:// 9) “Milestone Technology Provides
As a result, we are fully committed milestonesci.com/webcasts/. Cannabis Processor Quality and
to supporting the cannabis testing Efficiency in Terpene Extraction:
5) “Heavy Metals Determination in Medezin Labs, Case Study,”
industry for all their microwave di-
Cannabis Plant, Concentrates and https://milestonesci.com/
gestion and extraction needs as ex- Edibles Utilizing the Milestone cannabis-terpenes-extraction/.
emplified by these two client testi- Ethos UP,” Milestone in Technical
monials (8,9). Note, https://milestonesci.
com/cannabis-testing/.

References 6) “Simultaneous Mixed-Batch Sample Ryan Boyle and Eric Farrell

Preparation for Metals Testing are with Milestone Inc., in Shelton,
1) “Growing Your Cannabis Testing of Medical Cannabis Samples Connecticut. Direct correspondence
Capabilities,” Milestone Inc. Utilizing the Milestone UltraWAVE,” to: [email protected] and
Product Note, https://milestonesci. Milestone in Technical Note; https:// [email protected]
com/cannabis-testing/. milestonesci.com/cannabis-testing/.

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38 Feature

LC–MS/MS with ESI and APCI

Sources for Meeting California
Cannabis Pesticide and Mycotoxin
Residue Regulatory Requirements
Two different liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods with
electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were used for low-
level analysis of 72 pesticides (including the very hydrophobic and chlorinated pesticides analyzed
by gas chromatography [GC]–MS) in cannabis. The ability to screen and quantitate all 72 pesticides,
including the very hydrophobic and chlorinated GC–MS amenable pesticides, in cannabis with only
LC–MS/MS with ESI and APCI makes this approach a novel way of screening and quantitation of
pesticides in cannabis and different matrices with a single instrument

Avinash Dalmia, Erasmus Cudjoe, Toby Astill, Jacob Jalali, Jason P. Weisenseel,
Feng Qin, Molly Murphy, and Travis Ruthenberg

M
ore than half of the United States has legal- cannabis is an important consumer safety topic. Recent
ized the use of medical cannabis because of its news has reported an alarming percentage of cannabis
therapeutic benefi ts for ailments such as can- products to be tainted by high levels of pesticide resi-
CAPJAH/SHUTTERSTOCK.COM

cer, multiple sclerosis, and amyotrophic lateral sclerosis due, prompting recalls and public-safety alerts. Banned
(ALS or Lou Gehrig’s disease) (1–3). Like traditional ag- pesticides such as myclobutanil, imidacloprid, abamec-
riculture crops, pesticides are sometimes used in can- tin, etoxazole, and spiromesifen have been detected as
nabis cultivation to protect plants from pests and im- residues on cannabis flowers and concentrated further
prove growth yield. Chronic exposure to pesticides can in extracts and edibles. In Colorado, 20,000 packages of
pose serious health risks; therefore, pesticide analysis in cannabis flowers in October 2015 were recalled because

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 39

of pesticide contamination, and in the United States have developed MS LX50 ultrahigh-pressure liquid
November 2016, Oregon officials is- their own testing guidelines. Ore- chromatography (UHPLC) system,
sued a health alert for specific batch- gon was the first state to come up and detection was achieved using a
es of cannabis. Moreover, many of with comprehensive guidelines for PerkinElmer Q-Sight 220 MS/MS de-
today’s cannabis products are in- pesticide residue analysis in canna- tector with a dual ionization ESI and
haled after combustion, so there is bis (8) and set regulatory limits for APCI source; ESI and APCI operate
growing concern among consum- 59 pesticides in cannabis. Howev- independently with two separate
ers and regulators because of the er, California has issued more strin- inlets. Instrument control, data ac-
unknown effects of pesticide com- gent action limits for 66 pesticides quisition, and data processing were
pounds when they are inhaled (4,5). (including all but one of those found performed using Simplicity 3Q soft-
The growing conditions for cannabis on the Oregon state list, and eight ware (PerkinElmer).
are also conducive to the growth of more) and fi ve mycotoxins residues
molds and fungi, which can produce in cannabis flower and edibles (9). Sample Preparation Method
carcinogenic mycotoxins including Numerous reports for pesticide Below is the step-by-step sample
ochratoxin A and aflatoxins. As a re- analysis in cannabis have been pub- preparation procedure with 10-fold
sult, testing for the levels of pesti- lished but these studies have certain dilution:
cide and mycotoxins in cannabis is deficiencies (10–12). Most of these • Take approximately 5 g of
important to ensure consumer safe- studies either do not achieve de- cannabis flower as a representative
ty and quality control. tection limits to meet the California of each sample batch and grind
it finely using a grinder.
High performance liquid chroma- state action limits or use time-con-
tography–tandem mass spectrome- suming sample preparation meth- • Measure 1 g of sample and place
try (HPLC–MS/MS) has emerged as ods (for example, QuEChERS [quick, it into a 50-mL centrifuge tube.
the method of choice for pesticide easy, cheap, effective, rugged, and • Spike 10 μL of internal
and mycotoxin analysis because it safe] with dispersive solid-phase ex- standard solution.
offers superior selectivity, sensitiv- traction [dSPE]) with poor recover- • Add three steel balls (10 mm in
ity, ruggedness, and does not re- ies for some of the pesticides, which diameter) to the tube for efficient
quire extensive sample preparation require use of both LC–MS/MS- and extraction during vortex mixing.
before analysis. Although gas chro- GC–MS/MS-based instruments for • Add 5 mL of LC–MS grade
matography–tandem mass spec- analysis of all the pesticides. This re- acetonitrile to the tube and cap it.
trometry (GC–MS/MS) methods have quirement increases cost, complex- • Place the tube on multitube
been developed for pesticide analy- ity, and turnaround time of analysis vortex mixer and allow it
sis in cannabis samples, they are only substantially. In this work, 66 pes- to vortex for 10 min.
applicable to a smaller subset num- ticides (including very hydropho- • Centrifuge extract in tube
ber of analytes. Compounds such bic and chlorinated pesticides typ- for 10 min at 3000 rpm.
as daminozide, a highly polar com- ically analyzed by GC–MS/MS) and • Filter the solvent into a 5-mL
pound, and abamectin, a high-mo- fi ve mycotoxins spiked in canna- glass amber vial using a 0.22-μm
lecular-weight compound, are not bis flower extracts were analyzed at nylon syringe-filter and cap it.
amenable to analysis by GC–MS/MS levels well below the action limits • Label the bottle with the
because they are heat labile and de- specified by California. An LC–MS/ sample identification.
grade in either the GC injection port MS instrument was used with elec- • Transfer 0.5 mL of extracted
or the column at high temperature. trospray ionization (ESI) and atmos- sample into a 2-mL HPLC vial and
GC–MS/MS methods are not as ro- pheric pressure chemical ionization dilute it with 0.5 mL of LC–MS-
grade acetonitrile and mix it.
bust as LC–MS/MS methods for pes- (APCI) sources and a simple solvent
ticide analysis in complex matrices extraction method with excellent re- • Inject 3 μL of sample for
because they require extensive sam- coveries for all analytes in accepta- LC–MS/MS analysis, using
ple preparation to prevent GC injec- ble range of 70–120%. pesticide methods.
tion port contamination from com-
plex matrices (6,7). Experimental LC Method and
Because there is no federal guid- Hardware and Software MS Source Conditions
ance for the analysis of pesticides in Chromatographic separation was The LC method and MS source pa-
cannabis samples, different states in conducted on a PerkinElmer LC–MS/ rameters are shown in Table I.

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

40 Feature

Table I: LC method and MS source conditions

LC Conditions
LC column PerkinElmer Quasar Pesticide column (100 mm × 4.6 mm, 2.7 μm)
Mobile-phase A
2 mM ammonium formate + 0.1% formic acid (in water)
(ESI method)
Mobile-phase B
2 mM ammonium formate + 0.1% formic acid (in methanol)
(ESI method)
A 18.5 and 6 min LC method with gradient was used for the
Mobile-phase gradient
LC–MS/MS method with ESI and APCI source, respectively.
Column oven temperature 30 ºC
Auto sampler temperature 10 ºC
Injection volume 3.0 μL for LC–MS/MS method with ESI source. 10 μL for LC–MS/MS method with APCI source.
MS Source Conditions for ESI Source and APCI Source
ESI voltage (positive) +5500 V
ESI voltage (negative) -4200 V
APCI Corona Discharge -5 μA
Drying gas 120 arbitrary units
Nebulizer gas 350 arbitrary units
Source temperature 315 ºC
HSID temperature 200 ºC
Detection mode Time-managed MRM

Results and Discussion on the LC leads to poor chromato- with good recovery in a complex
Analytical Challenges for graphic peaks for early eluted polar cannabis matrix.
Testing Pesticide Residues compounds. To overcome this prob- Normally, the analysis of pesticides
in Cannabis Samples lem, a small sample injection volume in cannabis and other food matri-
Since the pesticides tested in this of 3 μL was used in this study. ces is done by both GC–MS/MS and
study included both polar and non- Pesticide analysis in cannabis is LC–MS/MS since some nonpolar and
polar compounds, 100% acetoni- very challenging since its matrix chlorinated pesticides are difficult to
trile was used to extract all the ana- composition is very complex and ionize with an electrospray ion source
lytes from the samples. Because of contains compounds from different (13,14). To demonstrate the conveni-
the cannabis matrix’s hydrophobici- classes such as cannabinoids, ter- ence of a single method, an LC–MS/
ty, further dilution of the extract was penes, hydrocarbons, sugars, fatty MS method was developed using both
performed with the aqueous mobile acids, flavonoids, and others. Sam- APCI and ESI techniques to analyze all
phase to make it compatible with the ple matrix effect remains the main the pesticides on the California regu-
reversed phase column. This protocol concern for LC–MS/MS, and leads lated pesticide list with the addition-
resulted in lower recoveries of some to variable signal ion suppression al benefits of improved throughput,
of the pesticides because of precipi- and matrix interference. Moreover, reduced complexity, and lower cost
tation. To achieve a higher performing quantification of pesticide residues of analysis. Typically, the dirty matrix
method, cannabis extracts were dilut- in cannabis is a difficult task because found with cannabis samples would
ed with acetonitrile by an overall fac- of the great disparity in high con- quickly foul the conventional GC–
tor of 10 to achieve high recovery of centration levels of naturally occur- MS/MS and LC–MS/MS systems and
pesticides and reduce matrix effects. ring cannabinoids as well as high ter- this contamination would increase the
However, the reversed-phase LC pene content. In this work, we used maintenance costs and downtime re-
method used aqueous mobile phase a generic extraction method with di- sulting in a loss of productivity. We
at the beginning of the LC run to help lution, selected the best multiple re- showed that the LC–MS/MS meth-
better retain the polar compounds on action monitoring (MRM) transitions od developed in this work would be
the column. Injecting an organic sol- and optimized the LC gradient to al- more immune to contamination from
vent, such as an acetonitrile sample, low low-level analysis of pesticides the dirty cannabis matrix.

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42 Feature

Table II: LOQs for California category II pesticides with LC–MS/MS in cannabis. Red/Green: pesticides typically analyzed by GC–MS/MS;
red/black: pesticides analyzed by LC–MS/MS with ESI; green: pesticides analyzed LC–MS/MS with APCI.

Category II Residual LOQ

S. No. Action Level (μg/g) Action Level/LOQ
Pesticide LC–MS/MS (μg/g) %CV (n=7)
1 Abamectin 0.025 10.6 0.1 4
2 Acephate 0.010 3.1 0.1 10
3 Acequinocyl 0.025 13.3 0.1 4
4 Acetamiprid 0.010 13.1 0.1 10
5 Azoxystrobin 0.005 5.0 0.1 20
6 Bifenazate 0.010 10.8 0.1 10
7 Bifenthrin 0.010 14.4 0.5 50
8 Boscalid 0.025 12.2 0.1 4
9 Captan 0.25 7.0 0.7 2.8
10 Carbaryl 0.010 9.5 0.5 50
11 Chlorantraniliprole 0.025 5.6 10.0 400
12 Clofentezine 0.010 11.3 0.1 10
13 Cyfluthrin 0.25 19.1 1.0 4
14 Cypermethrin 0.100 20.0 1.0 10
15 Diazinon 0.005 3.8 0.2 40
16 Dimethomorph 0.005 1.4 2.0 400
17 Etoxazole 0.005 13.5 0.1 20
18 Fenhexamid 0.010 12.5 0.1 10
19 Fenpyroximate 0.005 6.9 0.1 20
20 Flonicamid 0.010 10.2 0.1 10
21 Fludioxonil 0.050 9.5 0.1 2
22 Hexythiazox 0.005 8.4 0.1 20
23 Imidacloprid 0.010 10.3 3.0 300
24 Kresoxim-methyl 0.025 8.1 0.1 4
25 Malathion 0.010 14.7 0.5 50
26 Metalaxyl 0.010 8.0 2.0 200
27 Methomyl 0.010 8.5 0.1 10
28 Myclobutanil 0.010 10.4 0.1 10
29 Naled 0.010 8.4 0.1 10
30 Oxamyl 0.010 6.7 0.2 20
31 Pentachloronitrobenzene 0.010 13.0 0.1 10
32 Permethrin 0.010 16.0 0.5 50
33 Phosmet 0.005 13.3 0.1 20
34 Piperonylbutoxide 0.005 3.5 3.0 600
35 Prallethrin 0.025 7.4 0.1 4
36 Propiconazole 0.015 8.9 0.1 6.7
37 Pyrethrins 0.1 1.4 0.5 5
38 Pyridaben 0.010 7.9 0.1 10
39 Spinetoram 0.005 13.8 0.1 20
40 Spinosad 0.005 9.3 0.1 20
41 Spiromesifen 0.010 9.4 0.1 10
42 Spirotetramat 0.010 8.4 0.1 10
43 Tebuconazole 0.005 11.0 0.1 20
44 Thiamethoxam 0.010 3.6 4.5 450
45 Trifloxystrobin 0.005 8.4 0.1 20

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 43

Detectability and Reproducibility Table III: LOQs for California category II mycotoxins with LC–MS/MS in cannabis
Figure 1 shows MRM chromatograms
LOQ Action
with excellent signal-to-noise ratios S. Category II Action
LC–MS/ Level/
(S/N) for a representative set of pes- No. Mycotoxin %CV (n = 7) Level (μg/g)
LOQ
MS (μg/g)
ticides spiked at a level of 0.01 μg/g
in the cannabis flower. The limits of 1 Ochratoxin A 0.010 18 0.020 2.0
quantification (LOQs) and response 2 Aflatoxin B1 0.001 18 — —
reproducibility at the LOQ level for 3 Aflatoxin B2 0.0015 14 — —
each of the pesticides (category II 4 Aflatoxin G1 0.010 18 — —
and I) and mycotoxins in cannabis ex-
5 Aflatoxin G2 0.0015 19 — —
tract are summarized in Tables II–IV.
Aflatoxin
The LOQs were determined by con- 6 0.005 NA 0.020 4.0
(B1+B2+G1+G2)
sidering both the signals of the quan-
tifier and qualifier ions (S/N > 10 for
both) and ensuring that the product Table IV: LOQs for California category I pesticides with LC–MS/MS in cannabis. Red/
green: pesticides typically analyzed by GC–MS/MS; red/black: pesticides analyzed by
ion ratios were within the 20% toler-
LC–MS/MS with ESI; green: pesticides analyzed by LC–MS/MS with APCI.
ance windows of the expected ratio.
LC–MS/MS LOQ
As demonstrated in Tables II and III, Category I Action Level Action Level/
the LOQs determined in this study S. No. %CV
Residual Pesticide (μg/g) (μg/g) LOQ
are well below the California action (n = 7)

limit by a factor of 2 to 600 for all cat- 1 Aldicarb 0.010 10.6 0.1 10
egory II pesticides and mycotoxins 2 Carbofuran 0.010 3.1 0.1 10
listed. The response relative stand- 3 Chlordane 0.05 13.3 0.1 2
ard deviation (RSD) for each pesticide
4 Chlorfenpyr 0.05 13.1 0.1 2
and mycotoxin at its LOQ level in the
5 Chlorpyrifos 0.010 5.0 0.1 10
cannabis matrix was less than 20%.
6 Coumaphos 0.010 10.8 0.1 10
The retention time for each analyte
was reproducible within ±0.1 min over 7 Daminozide 0.015 14.4 0.1 6.67
a 24-h period. This demonstrates that 8 DDVP (dichlorvos) 0.025 12.2 0.1 4
the method is more than adequately 9 Dimethoate 0.010 3.8 0.1 10
sensitive and reproducible for pesti- 10 Ethoprop(hos) 0.010 9.5 0.1 10
cides and mycotoxins analysis in can- 11 Etofenprox 0.010 5.6 0.1 10
nabis at the regulatory limit specified
12 Fenoxycarb 0.010 11.3 0.1 10
by the state of California.
13 Fipronil 0.010 19.1 0.1 10

Sample Matrix-Matched 14 Imazalil 0.010 23.1 0.1 10

Calibration Standards 15 Methiocarb 0.010 3.8 0.1 10
Matrix-matched calibration is the 16 Methyl parathion 0.040 1.4 0.1 2.5
preferred analytical procedure for 17 Mevinphos 0.025 13.5 0.1 4
quantitation because it compen- 18 Paclobutrazol 0.010 12.5 0.1 10
sates for matrix effects that are
19 Propoxur 0.010 6.9 0.1 10
prevalent in cannabis samples. The
20 Spiroxamine 0.010 10.2 0.1 10
decrease or increase in response is
attributed to ion suppression of the 21 Thiacloprid 0.010 9.5 0.1 10

analytes during ionization by the

presence of coeluted matrix com- cannabis flower extract samples calibration fit R 2 of greater than 0.99
pounds. Because of sample matrix spiked with varying concentrations for all the analytes.
effects, a matrix matched calibra- of pesticides and mycotoxins over
tion curve was used for quantitation a range of 0.1–1000 ng/mL. The cal- Recovery Studies
and generated by injecting blank ibration curves for all pesticides with Solvent Extraction
cannabis flower extracts and blank and mycotoxins were linear with a The QuEChERS extraction technique

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

44 Feature

Table V: Recovery of category II pesticides at two different levels from cannabis with acetonitrile solvent extraction method

Category II Residual Low Level 0.1 μg/g High Level 1 μg/g

S.No.
Pesticide Recovery (%) RSD (%) (n=5) Recovery (%) RSD (%) (n=5)
1 Abamectin 85 10 89 9
2 Acephate 93 16 91 9
3 Acequinocyl 90 11 86 6
4 Acetamiprid 87 13 95 9
5 Azoxystrobin 87 12 92 8
6 Bifenazate 88 8 88 7
7 Bifenthrin 84 13 94 7
8 Boscalid 87 10 89 5
9 Captan — — 70 15
10 Carbaryl 84 12 92 10
11 Chlorantraniliprole 88 13 90 8
12 Clofentezine 87 13 91 12
13 Cyfluthrin — — 97 17
14 Cypermethrin 98 18 85 13
15 Diazinon 96 10 95 10
16 Dimethomorph 87 15 90 7
17 Etoxazole 89 10 92 10
18 Fenhexamid 87 12 87 7
19 Fenpyroximate 87 9 93 8
20 Flonicamid 93 15 92 12
21 Fludioxonil 94 13 93 8
22 Hexythiazox 86 11 93 7
23 Imidacloprid 89 11 91 9
24 Kresoxim-methyl 91 10 95 8
25 Malathion 90 12 91 7
26 Metalaxyl 86 10 92 8
27 Methomyl 89 10 90 9
28 Myclobutanil 84 10 93 7
29 Naled 87 10 91 7
30 Oxamyl 93 16 94 9
31 Pentachloronitrobenzene 80 16 88 8
32 Permethrin 87 17 92 9
33 Phosmet 86 11 91 7
34 Piperonylbutoxide 91 8 94 8
35 Prallethrin 88 15 94 8
36 Propiconazole 90 14 95 11
37 Pyrethrins 89 12 93 9
38 Pyridaben 84 13 92 9
39 Spinetoram 93 13 94 9
40 Spinosad 88 14 90 10
41 Spiromesifen 90 8 92 5
42 Spirotetramat 97 10 90 7
43 Tebuconazole 94 12 91 7
44 Thiamethoxam 90 10 95 10
45 Trifloxystrobin 86 12 93 9

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 45

is a common approach for the ex- Table VI: Recovery of category II mycotoxins at two different levels from cannabis with
acetonitrile solvent extraction method
traction of low levels of contaminants
such as pesticides from fruit and veg- Low Level 0.02 μg/g High Level 0.1 μg/g
S. Category II
etable matrices with higher water No. Mycotoxin RSD (%) Recovery RSD (%)
Recovery (%)
content (15). The method includes (n = 5) (%) (n = 5)
extraction of a broad range of pesti- 1 Aflatoxin B1 75 15 84 9
cides and removal of sugars, organ- 2 Aflatoxin B2 78 14 82 9
ic acids, and other compounds com-
3 Aflatoxin G1 76 12 85 7
monly found in fruits and vegetables
4 Aflatoxin G2 79 12 84 6
(16–20). It is not a suitable method for
very polar pesticides, such as dami- 5 Ochratoxin A 78 20 83 7

nozide, which are included in both

the California and other states reg-
Table VII: Recovery of category I pesticides at two different levels from cannabis with
ulatory framework. Daminozide is acetonitrile solvent extraction method
too polar to be extracted efficient-
Category Low Level 0.1 μg/g High Level 1 μg/g
ly with QuEChERS; it remains in the S.
I Residual Recovery RSD (%) Recovery RSD (%)
aqueous phase and does not parti- No.
Pesticide (%) (n = 5) (%) (n = 5)
tion into the organic solvent during
the salting out step. The recovery of 1 Aldicarb 87 11 94 11

daminozide from a cannabis matrix 2 Carbofuran 86 11 91 9

with QuEChERS extraction has been 3 Chlordane 87 19 92 10
reported to be less than 10% (10). 4 Chlorfenapyr 95 15 99 10
Moreover, a typical cannabis matrix 5 Chlorpyrifos 94 8 92 8
contains mostly hydrophobic com-
6 Coumaphos 90 12 95 10
pounds such as cannabinoids and
7 Daminozide 82 15 80 14
terpenes, and therefore the QuECh-
DDVP
ERS extraction method does not re- 8 94 14 91 11
(dichlorvos)
move the matrix interfering com-
9 Dimethoate 89 11 96 9
pounds during the salting out step.
Different groups have tried to devel- 10 Ethoprop(hos) 92 9 94 7

op an advanced QuEChERS method 11 Etofenprox 88 13 93 8

with a dSPE step that utilizes primary 12 Fenoxycarb 91 11 93 7
secondary amine (PSA) and other ad- 13 Fipronil 89 9 95 8
sorbents to remove matrix from can- 14 Imazalil 86 10 89 10
nabis extract. But the addition of the
15 Methiocarb 81 9 93 6
dSPE step to the QuEChERS method
Methyl
not only makes this method more la- 16 89 14 96 11
parathion
borious and expensive, but also leads
17 Mevinphos 86 10 95 10
to low recoveries of compounds such
18 Paclobutrazol 79 13 90 6
as spinosad, spirotetramat, spiorox-
amine, ochratoxin A, and a few oth- 19 Propoxur 91 13 93 9

ers (11,12). These low recoveries are a 20 Spiroxamine 88 9 89 9

result of these compounds binding to 21 Thiacloprid 89 13 95 10
the PSA adsorbent in the dSPE step.
In light of the above mentioned this method, fortified cannabis flower two levels (low and high) of all pesti-
shortcomings of the QuEChERS samples were used to determine pes- cide (0.1 and 1 μg/g) and mycotoxin
method for the extraction of pesti- ticides and mycotoxin recovery. The (0.02 and 0.1 μg/g) standards. These
cides from cannabis matrix, the ap- cannabis flower samples were tested two levels were chosen based on reg-
plication team used a simple ace- to confirm the absence of pesticides ulatory limits for pesticides and myco-
tonitrile-based solvent extraction before they were spiked. Five can- toxins in cannabis from California and
method for extraction. To confirm nabis flower samples were spiked at other states. Tables V–VII show that

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

46 Feature

values were not reported at the low

(a) (b) 7.09
spiked value since it was below their
3.81
10,000 LOQ value.
Intensity (cps)

Intensity (cps)
1000

500 5000
LC–MS/MS Method with Optimum
MRM Transitions for Challenging
0 0
3.5 4
Time (min)
4.5 6.8 7 7.2
Time (min)
7.4
Analytes in Cannabis Matrices
(c) (d) As stated, cannabis is a challenging
10,000
15,000 12.33 13.06
matrix to test, and this is compound-
Intensity (cps)
Intensity (cps)

10,000 ed by the low concentration level of

5000
the pesticides. To ensure the highest
5000
analytical confidence, multiple MRM
0
12 12.2 12.4 12.6
0
12.8 13 13.2 13.4 transitions for a number of pesticides
Time (min) Time (min)
with minimal matrix interference in
(e) 5.67 (f) 7.56
10,000 15,000
the cannabis matrix were determined
for low-level detection. For example,
Intensity (cps)
Intensity (cps)

10,000

5000 acequinocyl is an insecticide and can

5000
be ionized easily as a protonated mo-
0 0
lecular ion in a standard, but the MRM
5.5 5.6 5.7 5.8 5.9 6 6.1 6.2 7.2 7.4 7.6 7.8 8
Time (min) Time (min) transitions, based on protonated mo-
lecular ion in the cannabis matrix,
Figure 1: MRM chromatogram of representative set of pesticides: (a) oxamyl, (b) metal- showed a poor LOQ of 0.5–1 μg/g,
axyl, (c) fenpyroximate, (d) mycyclobutanil, (e) etofenprox, and (f) azoxystrobin spiked at a
level of 0.01 μg/g in cannabis matrix. about 5–10 times higher than its ac-
tion limit for the state of California.
Therefore, MRM transitions based on
alternative modes of ionization, such
14.59
(a)
as adduct formation, were deter-
20,000
mined to reduce matrix interference
and achieve an LOQ of 0.025 μg/g
Intensity (cps)

15,000

10,000
(four times below action limits) for
14.51
acequinocyl in the cannabis matrix.
5000
Figure 2 shows the signal overlay of
0
14.3 14.4 14.5 14.6 14.7 14.8 14.9
blank cannabis matrix and acequino-
Time (min)

cyl spiked at level of 0.1 μg/g in can-

(b) nabis with MRM transitions based on
25,000
protonated molecular ion and adduct
20,000
ion of acequinocyl. This figure dis-
Intensity (cps)

15,000
plays that optimum acequinocyl MRM
10,000 transitions helped in achieving lower
5000 detection limits because of minimal
0
matrix interference.
14.3 14.4 14.5 14.6 14.7 14.8 14.9
Time (min)
High molecular weight compounds
Figure 2: (a) Overlay of response of cannabis matrix (red) and acequinocyl (green) spiked such as abamectin, and some ear-
at 0.1 μg/g in cannabis matrix with MRM transition based on protonated molecular ion. ly eluting polar compounds, such as
(b) Overlay of response of cannabis matrix (red) and acequinocyl (green) spiked at
daminozide, are difficult to meas-
0.1 μg/g in cannabis matrix with MRM transition based on adduct ion.
ure at low levels using GC–MS/MS
since they decompose either in a
high-temperature GC injector or a
absolute recoveries of 66 pesticides range of 70–120% with RSD less than GC oven. Although high-molecular-
and five mycotoxins at two differ- 20% for five cannabis flower sam- weight compounds such as abamec-
ent levels were within the acceptable ples. For two pesticides, the recovery tin and polar compounds such as

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48 Feature

were set to maximize signals for high-

(a) 3.50E+05
3.00E+05
molecular-weight and polar pesti-
cides. Abamectin is also prone to
Abamectin signal

2.50E+05

2.00E+05
sodium and potassium adduct for-
1.50E+05

1.00E+05
mation from the sodium and potas-
5.00E+04 sium ions leached into mobile phase
0.00E+00
0 50 100 150 200 250 300 350 400 450 from glassware. Because it is difficult
Source temperature (ºC)
to control the amount of sodium and
(b) 5.00E+05
potassium ions leached from glass-
4.00E+05
Abamectin signal

3.00E+05
ware, the use of the sodium adduct
2.00E+05 for abamectin as Q1 (parent ion) mass
1.00E+05 for analysis would lead to response
0.00E+00
0 50 100 150 200 250 300 variation. To reduce sodium or potas-
HSID temperature (ºC)
sium adduct formation, a controlled
Figure 3: Abamectin signal as a function of (a) ESI source and (b) HSID temperature. amount of ammonium salt was add-
ed to the mobile phase. The combi-
nation of ammonium salt in the mo-
bile phase and optimum temperature
conditions resulted in good and re-
Pyrethrin | C21H28O3 Pyrethrin || C22H28O5
producible signals for abamectin.
O O O

O O O
O Analysis of Pesticides Typically
Jasmolin | C21H30O3 Jasmolin || C22H30O5
Analyzed by GC–MS/MS
O O O
O
by LC–MS/MS
O O O

Cinerin | C20H28O3
O A number of pesticides in cannabis,
Cinerin || C21H28O5
regulated by California and other
O
O O states, are traditionally analyzed us-
O O O
O O ing GC–MS/MS with an electron ion-
ization source since these pesticides
Figure 4: Structure of six isomers of pyrethrins.
have low proton affinity, which results
in low ionization efficiency with the
ESI source. Some examples of these
(a) (b) pesticides analyzed normally ana-
lyzed with GC–MS are cypermethrin,
CI CI
NO2 cyfluthrin, captan, naled, permethrin,

CI and pyrethrins. To achieve the re-

CI CI CI quired sensitivity, the selected MRMs
were optimized with a heated electro-
spray source. LOQ for these analytes
CI CI CI CI CI were in the range of 0.01–0.25 μg/g,

CI CI which was well below the California

action limits.

Analysis of Pyrethrins
Figure 5: Structure of (a) pentachloronitrobenzene and (b) chlordane.
Isomers in Cannabis
The pyrethrins are a class of organ-
daminozide can be ionized with the desolvation (HSID) or heated inter- ic compounds normally derived from
ESI source, they are also prone to de- face temperature and source tem- Chrysanthemum cinerariifolium that
composition at high temperatures. perature. Based on these results, the have potent insecticidal activity by
Figure 3 shows abamectin response optimum temperature values for the targeting the nervous systems of in-
as a function of hot-surface induced ESI source and HSID temperature sects. Pyrethrins are a group of six

CANNABIS SCIENCE AND TECHNOLOGY | www.CannabisScienceTech.com VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018

 Featture 49

isomers and their structures are dis-

played in Figure 4. The naturally oc-
curring pyrethrins, extracted from 2000

chrysanthemum flowers, are esters 1500

Intensity (cps)
of chrysanthemic acid (pyrethrin I, ci-
1000
nerin I, and jasmolin I) and esters of
pyrethric acid (pyrethrin II, cinerin II, 500

and jasmolin II). In the United States, 0

0 1 2 3 4 5
the pyrethrum extract is standardized Time (min)

as 45–55% w/w total pyrethrins and

in a commercially available pyrethrin Figure 6: Sample chromatogram of pentachloronitrobenzene (PCNB) spiked at level of
0.1 μg/g in a cannabis matrix using LC–MS/MS system with APCI source.
standard, the percentage of pyrethrin
I, pyrethrin II, cinerin I, cinerin II, jas-
molin I, and jasmolin II is about 56.1%,
0.50
27.8%, 5.7%, 3.8%, 4%, and 2.6%, re-
100 ppb Diazinon peak area ratio to internal

spectively. A number of compounds 0.45

in cannabis mimic the structure of py- 0.40

RSD = 1.58%
standard in cannabis extract

rethrins, and therefore the analysis 0.35

of pyrethrins in cannabis is very dif- 0.30

ficult because of matrix interference. 0.25

The optimum MRM transitions and 0.20

LC gradient were developed to ana- 0.15

lyze the six pyrethrins at low levels in 0.10
the cannabis matrix with minimal ma- 0.05
trix interference. The LOQs obtained
0.00
with the LC–MS/MS method using 0 50 100 150 200 250 300 350 400 450 500
Run number
optimum MRM transitions and LC
gradient for six pyrethrins (pyrethrin I, Figure 7: Long term stability data over one week of injections of diazinon at a level of
pyrethrin II, cinerin I, cinerin II, jasmo- 100 ng/mL spiked in cannabis flower matrix extract.
lin 1, and jasmolin II) were 0.1, 0.1, 0.01,
0.03, 0.025, and 0.01 μg/g, respective- proton affinity and is therefore diffi- source, the LOQs of pentachloron-
ly, in cannabis flowers. cult to ionize efficiently with an ESI itrobenzene, chlordane, and chlor-
source. Because an APCI ion source fenapyr in cannabis were 0.01, 0.05,
Pesticides That Don’t Ionize Effec- is better suited for ionization of very and 0.05 μg/g, respectively.
tively with ESI Analyzed with APCI hydrophobic and nonpolar analytes,
Hydrophobic and halogenated pes- APCI was used to determine the de- Long-Term Stability Data with
ticides (for example, pentachloroni- tection limits of pentachloronitroben- Self-Cleaning Source in LC–MS/MS
trobenzene and chlordane) are tradi- zene and chlordane in cannabis. Also, Long-term stability data for pesticide
tionally analyzed by GC–MS/MS since the APCI ion source was used for low- and mycotoxin analysis in cannabis
they do not ionize effectively by LC– level analysis of chlorfenapyr in canna- samples was collected using an LC–
MS/MS with an ESI source. For refer- bis, since limits of detection for chlor- MS/MS system, fitted with ESI and
ence, the structure of the chlorinated fenapyr were improved by a factor of APCI sources, and combined with a
pesticides is shown in Figure 5. Since two with the APCI source in compari- heated and self-cleaning source with
pentachloronitrobenzene (PCNB) son to ESI source because of less ion a laminar flow interface. Figure 7
does not contain either hydrogen at- suppression. Figure 6 shows an excel- shows long-term response and sta-
oms, for loss of protons, or functional lent signal-to-noise ratio (S/N ⩾ 100) bility of the method for 100 ng/mL of
groups with either high proton affini- for PCNB spiked at level of 0.1 μg/g diazinon spiked in cannabis extract
ty or that can form ammonia or sodi- in the cannabis matrix using an LC– over one week. Long-term stability
um adducts, it cannot be ionized with MS/MS system with an APCI source. data for pesticide analysis in canna-
the ESI source. Similarly, chlordane is Using a fast 6-min LC–MS/MS meth- bis showed that response RSD over
highly chlorinated and has very low od with a short LC gradient and APCI one week for most of the pesticides

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

50 Feature

14) United States Department of

and mycotoxins was 1.5–20%. These References Agriculture Food Safety and
results demonstrated that the heat- 1) A.A. Monte, R.D. Zane, and K. Inspection Service, Office of
ed self-cleaning source in the LC– J. Heard, JAMA, J. Am. Med. Public Health Science,”Screening
MS/MS system would reduce main- Assoc. 313(3), 241–242 (2015). for Pesticides by LC/MS/MS and
GC/MS/MS,” 2018, available
tenance needs that are usually 2) J.C. Raber, S. Eizinga, and from https://www.fsis.usda.gov/
prevalent with this matrix. Most of C. Kaplan, J. Toxicol. Sci. wps/wcm/connect/499a8e9e-
the LC–MS/MS methods published in 40(6), 797–803 (2015). 49bd-480a-b8b6-d1867f96c39d/
the literature do not show long term CLG-PST5.pdf?MOD=AJPERES.
3) D. Stone, Regul. Toxicol.
stability data or state that they have Pharmacol. 69(3), 284–288 (2014). 15) M. Anastassiades, S.J. Lehotay,
to clean the electrospray source fre- D. Stajnbaher, and F.J. Schenk, J.
4) E. Mcdonough, “Tainted: The
AOAC Int. 86(2), 412–431 (2003).
quently to maintain the sensitivity of Problem With Pot and Pesticides,”
the mass spectrometer (21). Also, they High Times (2017), https:// 16) S.W.C. Chung and B.T.P.
hightimes.com/grow/tainted-the- Chan, J. Chromatogr. A
divert the LC flow to waste for the first
problem-with-pot-and-pesticides. 1217, 4815–4824 (2010).
few minutes (and after the last peak
is eluted) to reduce contamination 5) A. Lozano, “Pesticides in Marijuana 17) S.C. Cunha, S.J. Lehotay, K.
Pose a Growing Problem for Mastovska, J.O. Fernandes, M.
from unretained and late eluted ma- Cannabis Consumers,” LA Weekly Beatriz, and P.P. Oliveria, J. Sep.
trix compounds. In this study, excel- (2016), http://www.laweekly.com/ Sci. 30(4), 620–626 (2007).
lent long-term stability data was ob- news/pesticides-in-marijuana-
pose-a-growing-problem-for- 18) Y. Sapozhinikova, J. Agric. Food
tained without diverting the LC flow Chem. 62, 3684–3689 (2014).
cannabis-consumers-7526808.
from the MS in the first few minutes,
6) http://american-safe- 19) J. Wang and W. Cheung, J. AOAC
at the end of run, and without period-
access.s3.amazonaws.com/ Int. 99(2), 539–557 (2016).
ical cleaning of ion sources.
documents/AHP_ Cannabis_ 20) M. Villar-Pulido, B. Gilbert-
Monograph_Preview.pdf. Lopez, J.F. Garcia Reyes, N.R.
Conclusions Martos, and A. Molina-Diaz,
7) https://www.aocs.org/stay-
This study demonstrates a unique, informed/read-inform/featured- Talanta 85, 1419–1427 (2011).
quantitative, rapid, and reliable LC– articles/ the-highs-and-lows-of- 21) L. Geis-Asteggiante, S.J. Lehotay,
MS/MS method for analysis of differ- cannabis-testing-october-2016. R.A. Lightfield, T. Dutko, C. Ng,
ent cannabis pesticide and mycotox- 8) Exhibit A, Table 3. Pesticide and L. Bluhm, J. Chromatogr.
in residues in cannabis samples. The analytes and their action levels. A 1258, 43–54 (2012).
proposed solvent extraction meth- Oregon Administrative Rules
333-007-0400; Oregon/gov/
od is suitable for laboratories wanting
oha, effective 5/31/2017.
to comply with the state of California
regulations, because the recovery of 9) Chapter 5. Testing Laboratories
Section 5313 Residual Pesticides,
all pesticides and mycotoxins from a Bureau of Marijuana Control
cannabis matrix was in the accepta- Proposed Text of Regulations,
ble range of 70–120% with RSD less CA Code of Regulations,
than 20%. This method allowed iden- Title 16, 42, pp 23–26.
tification and quantification of 66 pes- 10) K.K. Stenerson and G. Oden,
ticides and five mycotoxins at low lev- Cannabis Science and
Technlogy 1(1), 48–53 (2018).
els (0.005 to 0.25 μg/g), which is well
below the actions limits set by the 11) J. Kowlaski, J.H. Dahl, A. Rigdon,
state of California with good preci- J. Cochran, D. Laine, and G. Avinash Dalmia and Jason P.
Fagras, Advancing the Analysis Weisenseel are with PerkinElmer, Inc.,
sion. The ability to screen and quan- of Medical Cannabis, supplement in Shelton, Connecticut. Erasmus
titate all 66 pesticides, including the to LCGC North Am. and Cudjoe, Toby Astill, and Feng Qin are
very hydrophobic and chlorinated Spectroscopy 35(5), 8–22 (2017). with PerkinElmer, Inc., in Woodbridge,
Ontario, Canada. Jacob Jalali is with
compounds normally analyzed on a 12) X. Wang, D. Mackowsky, J. PerkinElmer, Inc., in San Jose, Cali-
GC–MS/MS system, and the five my- Searfoss, and M. Telepchak, LCGC fornia. Molly Murphy is with SC Labs
cotoxins, makes this method a nov- North Am. 34(10), 20–27 (2016). in Tigard, Oregon. Travis Ruthen-
berg is with SC Labs in Santa Cruz,
el way to screen and quantitate pes- 13) L. Alder, K. Greulich, G. Kempe, California. Direct correspondence to:
ticides and mycotoxins in cannabis and B. Vieth, Mass. Spec. [email protected]
with a single instrument. Rev. 25, 838–865 (2006).

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 Canna
abis Crossroa
ads 51

The Cannabis Science Conference

Grows to New Levels
Conference organizer Joshua Crossney provides a brief overview of the key take away messages
presented at the 2018 Cannabis Science Conference.

Joshua Crossney

T
he 2018 Cannabis Science cannabis science in an impactful way. ence Conference congress: Rather than
Conference commenced with Dr. Meiri described recent advances in focus on a cure for cancer, we should
a sold out Canna Boot Camp profiling the phytocannabinoid compo- be looking at the causes of cancer
at Chalice Farms on Monday, August sitions of differing cannabis strain vari- and work to systematically eliminate
27, which was hosted by 13 compa- eties that are used for clinical purposes, them. She explained how causation of
nies. Attendees rotated through fi ve and also explained how this diversity of chronic, long term inflammation is the
zones, covering everything from cul- compounds affects the outcomes of name of the game, stating that how we
tivation and genomics to milling, ex- cannabis-based treatments. live equals how we feel. We must each
tractions, sample preparation, anal- Attendees compared notes on work to eliminate the toxins by detoxi-
ysis, and edibles manufacturing. “must-see” sessions and split their fying the environments that we live in,
Participants were thrilled to see ac- time between presentations within starting with our homes.
tor and comedian Jim Belushi work- the analytical, medical, and cultivation Drescher also explained that the
ing alongside Dr. Sue Sisley at the tracks. Presentation after presentation cause of cannabis prohibition was big
Boot Camp. Dr. Scott Kuzdzal, Vice and panel after panel, the excitement business greed that went to Washing-
President of Marketing at Shimadzu, level and information sharing grew to ton with deep pockets to protect their
elaborated on this year’s preconfer- a very powerful buzz that permeat- petroleum and plastic based interests.
ence workshop, saying it was a huge ed the meeting rooms and exhibition “All these years that we could have
success. “The Canna Boot Camp has floor. “There was an amazing energy been evaluating the efficacy of canna-
grown so large that all of us vendors level at this year’s show, a coming to- bis as a medicinal got lost, and now,
are already considering new oppor- gether of medical, analytical, and can- only now, can we begin to really start
tunities to improve the event for nabis experts that felt like an extend- understanding the versatility and the
next year, including travel to a can- ed cannabis family,” said Tracy Ryan, miracle of this plant,” she said.
nabis cultivation site and optional a keynote speaker from the medical Drescher’s final message was clear:
tracks on analytical techniques with track. Ryan’s talk delved into her jour- We as consumers ultimately have great
a greater focus on education, not ney from mother to CEO of Cannakids power and we are at a great precipice
demonstrations,” he said. and SavingSophie.org. of a new dawn in human history. This
is a new frontier, and everybody wants
Highlights from the Conference Plenary Address in on the big, gold rush in cannabis.
On the opening day of the conference, On Tuesday afternoon, the analytical “Wherever there is opportunity, there
throngs of attendees amassed at the track room filled for a phenomenal are opportunists. Where there is capi-
registration counters and gathered for plenary address entitled, “My Journey tal, there’s capitalists,” she explained.
OLEH MARKOV/SHUTTERSTOCK.COM

the analytical track’s keynote address from Cancer to Cannabis” by television “And where there is greed, there is ir-
by Dr. Dedi Meiri of the Technion Insti- and film star Fran Drescher. Drescher responsibility, there is a lack of vision,
tute in Israel. This keynote, as well as explained her journey pre-stardom, there is lack of planning, there is lack
the one delivered by Dr. Ethan Russo through her celebrity with The Nanny of respect for the user, the plant, and
of the International Cannabis and Can- to her battle with, and defeating of, the planet. And if how we live equals
nabinoids Institute on Wednesday, set uterine cancer. As she told her story, how we feel, and this plant can help
a tone of inspiration and accomplish- she quickly came to key messages that support our bodies to feel better, then
ment that together we are advancing resonated with the entire Cannabis Sci- it is up to us to ensure that this plant

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

52 Cannabis Crosssroads

is organic, grown in healthy soil, un- shared by professional athletes in the the “International Cannabis Updates”
der the sun with the fresh air—a hap- always popular, “Doc & Jocks” panel panel, hosted by Sharlene Mavor, Di-
py, healthy, organic plant!” moderated by Dr. Uma Dhanabalan. rector of the nonprofit Medical Canna-
A lot of people are compromis- With approximately 3000 attend- bis Research Australia, with panelists
ing that vision for a healthy plant, and ees, 150 companies, and 90+ poster Sandra Paola Santander Gonzalez,
Drescher declared that it is up to us, the presentations on the exhibit floor, the PhD, (Colombia), Dedi Meiri, PhD, (Is-
customer, to make sure that what we ask exhibit hall reached maximum den- rael), Paul Mavor, B. Pharm, (Australia),
for, what we buy, what we grow, what sity during the Tuesday evening mix- Dr. Ellen Campbell Grizzle, (Jamaica),
we manufacture, and what we present er sponsored by Shimadzu. It was a and Wolfgang Simon (Germany). Med-
to the American public is not echoing dream come true to see cannabis ex- ical Track keynotes were delivered by
the mistakes of the industrialists of the perts, medical professionals, ana- Bonni Goldstein, MD, (Medical Direc-
last century. We must all see ourselves lytical scientists, as well as patients, tor, Canna Centers) and Tracy Ryan
in this bigger responsibility and that professional athletes, and curious nov- (CEO & Founder, CannaKids and Sav-
how you live equals how you feel. “This ices come together to advance can- ing Sophie).
is too special a moment, this is too pre- nabis science. It was refreshing to see A highlight of the Medical Track
cious a gift from God—a miracle plant,” so much emphasis on pushing the was a presentation by Rylie Maedler
she said. “Don’t disrespect it, don’t dis- boundaries of cannabis science, and of Rylie’s Smile Foundation titled “A
respect the planet, don’t disrespect the to hear about so many people collab- Pediatric Medical Patient’s Fight to
planet by going low, go high!” orating in new ways. Live Normal.” Wanting other children
There were many great talks in the and herself to have legal access to this
Session Highlights cultivation track, including a very enter- healing plant, Maedler fought for le-
There were too many highlights to sum- taining presentation on “Bio Harmonic galization of pediatric medical canna-
marize here. But some of my personal Cultivation: Growing with Therapeu- bis in Delaware. Her perspective on
favorite sessions from the analytical tic Intention” delivered by John East- a child’s rights has since helped her
track were Dr. Sue Sisley’s (Scottsdale erling of Happy Tree Microbes. “Ama- change several more laws that give
Research Institute) moving presenta- zon John” as he is known to his friends, better access for pediatric patients.
tion entitled, “From Pills to Pot: Data detailed how he transitioned from an Hearing Maedler’s story and viewpoint
on Cannabis for Chronic Pain and Use adventurous, young treasure seeker gave all attendees a new perspective
by Professional Athletes.” Dr. Sisley in Peru to developing an understand- on a pediatric cannabis patient’s fight.
explained how cannabis can help pro- ing of harmonic fields in the grow envi-
fessional athletes escape the horren- ronment, with Amazonian botanicals to Summary
dous jaws of prescription opiates. Dr. educate bacteria in the soil and a focus In closing, I’d like to thank everyone
Ethan Russo delivered a tour de force, on allowing the plant to express itself involved in making the 2018 Cannabis
educational keynote titled “Making at its fullest therapeutic value. Science Conference a success. Com-
Cannabis Better and Safer.” Reggie There were also many other great ing together was a beginning, keeping
Gaudino, PhD, (Steep Hill) delivered surprises, including a specially add- together is progress, working together
an overview of the various gene net- ed closing cultivation keynote by Ed is success, and growing together is
works associated with cannabinoid Rosenthal that described what may our mission! CSC 2019 will be held
and terpene production, explaining very well be the future of cannabis cul- from September 4–6 at the Oregon
the current status of the understand- tivation—a landrace Moroccan variety Convention Center in Portland.
ing of these networks. Furthermore, common in the Rif Mountains of North
several expert technical presentations Africa that grows only a single stem and About the Columnist
OLEH MARKOV/SHUTTERSTOCK.COM

from speakers representing the Uni- could be used in a breeding program Joshua Crossney is the
versity of Texas at Arlington, Columbia to develop plants especially adapted columnist and editor of
“Cannabis Crossroads”
Food Labs, Advion, Shimadzu, Eden for the “sea of green” method.
and a contributing editor
Labs, Phylos Bioscience, the Maryland The medical track also featured to Cannabis Science and
Medical Cannabis Commission, Agi- many amazing moments, including Technology magazine.
Crossney is also the president and
lent, PerkinElmer, and Millipore Sigma panels devoted to patient perspec-
CEO of CSC Events. Direct correspon-
were balanced with interactive panel tives, including one focused on treat- dence to: [email protected]
discussions and emotional stories ing pediatric patients, and a return of

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ADVERTISEMENT Ap
pplication Note 53

Adding New Synthetic Cannabinoids

to an Existing LC–MS/MS Method
Sharon Lupo and Frances Carroll, Restek Corporation

The analysis of synthetic cannabinoids and their metabolites can be a difficult task, one that is further
complicated by the ever-growing list of synthetic cannabinoids produced by illicit drugmakers. As shown
here, the retention and selectivity of the Raptor Biphenyl column allows new drugs to be added to an
existing method, providing labs with an important vehicle for improving efficiency and productivity.

When developing methods for synthetic cannabinoids JWH-073 isobars, as well as good separation of all tar-
and their metabolites, optimization of analysis time, res- get analytes from matrix interferences in urine. In the ev-
olution of isobaric compounds, method robustness, and er-changing landscape of illegal drugs, the ability to add
the ability to add emerging compounds are of ultimate im- emerging drugs to existing methods allows for much more
portance as they influence method effectiveness and lon- effective use of laboratory resources as analysts and in-
gevity. Because the Raptor Biphenyl column combines the struments can focus on routine sample analysis rather than
speed of superficially porous particles (SPP) with the res- new method development.
olution of highly selective USLC technology, we used it to
develop a simple, dilute-and-shoot method for synthet- Experimental Conditions
ic cannabinoids and their metabolites in urine. Our origi- For the existing method, samples were prepared at 5 ng/mL
nal work produced a fast method that separated all target in human urine and diluted 3x in a 0.2 μm PVDF Thom-
compounds in less than 7 min. son SINGLE StEP filter vial with water:methanol (50:50) pri-
Here, we expand on our original method and show or to analysis. The additional compounds were prepared
proof of concept for how new drugs can be added, while at 50 ng/mL in urine and also were diluted 3x and filtered
still maintaining complete resolution of JWH-018 and using the same technique. Analyses were performed on a

m/z 328155

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

m/z 344155

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

m/z 358155

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

Time (min)

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Time (min)

Figure 1: Original method developed for the combined analysis of synthetic cannabinoids and metabolites in diluted human urine.

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54 Application
n Note ADVERTISEMENT

Table I: Figure 1 analyte identification, retention times, and ion transitions

Figure 1 Peak Identifications tR (min) Precursor Ion Quantifier Product Ion Qualifier Product Ion
1. Pravadoline 2.15 379.29 135.04 114.16
2. AM2233 2.44 459.25 112.2 98.15
3. JWH-200-d5 2.47 390.34 155.07 NA
4. JWH-200 2.48 385.28 155.07 114.16
5. WIN 55, 212 3.34 427.29 155.07 127.14
6. JWH-073 N-butanoic acid 3.39 358.27 155.08 127.11
7. JWH-073 4-hydroxybutyl 3.4 344.24 155.09 127.09
8. JWH-018 N-pentanoic acid 3.49 372.18 155.08 127.14
9. JWH-018 5-hydroxypentyl-d5 3.54 363.5 155.08 NA
10. JWH-018 5-hydroxypentyl 3.55 358.27 155.08 127.11
11. JWH-073 6-hydroxyindole 3.77 344.24 155.09 127.09
12. JWH-073 5-hydroxyindole-d7 3.81 351.21 155.07 NA
13. JWH-073 5-hydroxyindole 3.83 344.24 155.09 127.09
14. JWH-073 7-hydroxyindole 3.92 344.24 155.09 127.09
15. JWH-018 6-hydroxyindole 3.94 358.27 155.08 127.11
16. JWH-018 5-hydroxyindole 3.99 358.27 155.08 127.11
17. JWH-018 7-hydroxyindole 4.08 358.27 155.08 127.11
18. RCS-4 4.15 322.27 135.12 77.09
19. XLR-11 4.21 330.25 232.17 125.1
20. JWH-015-d7 4.27 335.28 155.07 NA
21. JWH-250 4.27 336.28 121.12 91.07
22. JWH-015 4.29 328.26 155.07 127.13
23. AM2201 4.30 360.26 155.07 127.14
24. JWH-203 4.39 340.23 188.18 125.09
25. JWH-073 4.42 328.26 155.07 127.13
26. UR-144 4.44 312.32 214.17 125.1
27. JWH-073 4-hydroxyindole 4.53 344.24 155.09 127.09
28. JWH-018-d9 4.55 351.34 155.07 NA
29. JWH-018 4.57 342.27 155.08 127.11
30. JWH-081 4.64 372.28 185.12 157.09
31. JWH-018 4-hydroxyindole 4.66 358.27 155.08 127.11
32. JWH-122 4.69 356.29 169.12 141.11
33. JWH-019 4.70 356.29 155.07 127.1
34. JWH-210 4.84 370.31 183.12 153.26

Table II: Figure 2 analyte identification, retention times, and ion transitions

Figure 2 Peak Identifications tR (min) Precursor Ion Quantifier Product Ion Qualifier Product Ion

1. AB-FUBINACA 3.15 369.0 253.0 109.1

2. AB-PINACA 3.23 331.2 215.0 286.1
3. Salvinorin A 3.39 433.1 373.0 91.1
4. 5F-PB-22 4.04 377.2 232.1 143.9
5. PB-22 4.30 359.2 214.0 144.0
6. APINACA (AKB-48) 4.76 366.3 135.1 93.1

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ADVERTISEMENT Ap
pplication Note 55

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Time (min)

Figure 2: Overlay of new drugs (numbered peaks) and existing method chromatogram from Figure 1.

Waters ACQUITY UPLC I-Class equipped with a Xevo TQ-S cannabinoids and salvinorin A (a psychotropic terpenoid)
and a Shimadzu Nexera UHPLC equipped with a SCIEX API were analyzed under conditions identical to the established
4500 MS/MS. Instrument conditions were as follows, and method. As shown in Figure 2, which is an overlay of the
analyte transitions for the original analytes and the new new compounds (numbered peaks) and the existing sep-
drugs are provided in Tables I and II, respectively. aration from Figure 1 (unnumbered peaks), all compounds
Analytical column: Raptor Biphenyl (2.7 μm, 50 mm x were separated from early-eluting matrix interferences as
3.0 mm; cat.# 9309A5E) well as from each other without the need for adjustments to
Guard column: Raptor Biphenyl EXP guard column the mobile phase, gradient, or analytical column.
(2.7 μm, 5 mm x 3.0 mm; cat.# 9309A0253)
Mobile phase A: 0.1% Formic acid in water Conclusions
Mobile phase B: 0.1% Formic acid in acetonitrile Labs analyzing synthetic cannabinoids are under in-
Gradient: Time (min) %B creasing pressure to add new compounds to their ana-
0.00 25 lytical testing services. While this can be done through
1.00 25 new method development, adding new compounds to
5.00 95 an existing method can save time and resources. The
5.50 95 method shown here demonstrates the advantages of
5.51 25 the Raptor Biphenyl SPP column for this analysis: due to
7.00 25 the column’s highly retentive, selective characteristics,
Flow rate: 0.6 mL/min 35 drugs (including isomers and emerging compounds)
Injection volume: 2 μL can be analyzed quickly and effectively.
Column temp.: 30 °C
Ion mode: Positive ESI

Results
Chromatographic results for the original method are
presented in Figure 1. Complete resolution of all isobar-
ic compounds and good separation from major matrix
interferences were achieved with a cycle time of 5 min Restek Corporation
and a total analysis time of 7 min because of the unique 110 Benner Circle, Bellefonte, PA 16823
selectivity and retention of the Raptor Biphenyl column. tel. (814) 353-1300
To determine whether new drugs could be successful- website: www.restek.com
ly added to the original method, five additional synthetic

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56 Application
n Note ADVERTISEMENT

Analysis of Pesticide Residues, Mycotoxins, and

Potency in Cannabis Using QuEChERS Extraction,
ChloroFiltr® dSPE Cleanup, and LC–MS/MS
Brian Kinsella, Tina Fanning, and Xiaoyan Wang, UCT

This application note outlines a QuEChERS method for the simultaneous analysis of cannabis for 47
pesticides, five mycotoxins, and three cannabinoids.

Sample purification is carried out using UCT’s new cleanup

product SpinFiltr®. This product aims to combine the conven-
ience of classical dispersive-SPE (dSPE) with an ultrafiltration
tube containing a 0.2 μm filter membrane to simultaneous-
ly remove unwanted matrix components and filter the sam-
ple prior to LC or GC analysis. The SpinFiltr® dSPE tube fea-
tures PSA, C18, and ChloroFiltr® sorbents for sample cleanup.
ChloroFiltr® is a unique polymeric sorbent designed for the
removal of chlorophyll and unlike graphitized carbon black
(GCB), does not result in the loss of planar analytes. Liquid Figure 1: SpinFiltr®.
chromatography, using a Selectra® Aqueous C18 column,
coupled to tandem mass spectrometry (LC–MS/MS) is used
for analysis of the pesticides, mycotoxins, and cannabinoids. Results
Quantitation was performed against a six-point matrix-
Experimental Conditions matched calibration curve prepared in blank cannabis ex-
QuEChERS Procedure tract. With the exception of thiabendazole, no internal
Sample Extraction standards were used for quantitation.
1. Weigh 1 g of cannabis sample into a 50 mL
polypropylene centrifuge tube. Conclusions
2. Add internal standards. The use of SpinFiltr ® dSPE clean-up resulted in a puri-
3. Add 10 mL ultrapure water, vortex briefly, fied/filtered final extract. Chlorofiltr ® sorbent was used
and allow sample to hydrate for 15 min to selectively remove chlorophyll without compromising
(improves extraction efficiency). any planar compounds. Analysis of the samples was per-
4. Add 10 mL acetonitrile containing 2% formic acid. formed by LC–MS/MS utilizing a Selectra® Aqueous C18
5. Add the contents of the ECMSSC-MP Mylar HPLC column, which allowed for improved retention of the
pouch and shake for a minimum of 5 min (by more polar pesticides included in the method. The meth-
hand or mechanically). For this work, a Spex od was evaluated by fortifying cannabis samples with each
2010 Geno/Grinder ® was used (1500 RPM).
compound at four varying concentrations (n = 4 each).
6. Centrifuge the sample at ≥ 3000 × g for 5 min. The average recovery obtained was predominantly in the
range of 70–100% and the reproducibility ≤10%.
For Cannabinoid Analysis
1. Transfer appropriate amount of supernatant to autosa-
mpler vial for dilution

For Pesticide and Mycotoxin Analysis

SpinFiltr® dSPE Clean-up
1. Transfer 1mL of supernatant to a SpinFiltr ® dSPE clean-up
tube (ECQUSF54CT).
2. Vortex the sample for 30 s. UCT
3. Centrifuge the sample at ≥ 3000 × g for 5 min. 2731 Bartram Road, Bristol, PA 19007
4. Transfer the purified and filtered sample extract into an tel. (215) 781-9255; fax (215) 785-1226
autosampler vial for analysis. website: www.unitedchem.com

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 Cann
nabis Scien
nce Conferrence Product Profi
files 57

Microwave Digestion Purpose-Built LIMS for

Cannabis Testing Labs
The CEM MARS 6 is the highest performance micro-
wave digestion system on the market today. Capable of LabVantage Cannabis, a purpose-built LIMS platform,
digesting even the most challenging samples for trace accelerates deployment, lowering total cost and risk,
metals analysis, the MARS 6 is the clear solution for while delivering functionality critical to a compliant, ef-
every metals laboratory. Every system has hundreds of ficient cannabis testing lab. It’s easily configured—with-
preprogrammed methods and vessel options for high out needing to write a line of code—to stay current with
throughput and difficult samples. Add the world’s only fast-evolving regulations and business needs.
contactless in-situ temperature measurement system,
that accurately measures
Hosted on-premise or
the temperature of eve-
on the Cloud and availa-
ry sample and eliminates
ble through a perpetual
the need for probes, and
or SaaS license, LabVan-
you have a microwave
tage Cannabis features
that takes the guesswork
American Herbal Pharma-
out of digestion. With
copoeia® tests for canna-
MARS 6 you get bet-
bis inforescense, a grower
ter digestions for better
portal for remote sample
analyses.
requests, COAs custom-
ized for local regulations,
includes an interface with
METRC, and supports ISO/IEC 17025, ISO 9001:2015,
GLP, and 21 CFR Part 11.

CEM Corporation LabVantage Solutions

Matthews, NC Somerset, NJ
www.cem.com www.labvantage.com

Nitrogen Lab Server: N250M/N400M Sorbtech High Quality Adsorbents

Proton OnSite’s Nitrogen Running your laboratory requires deadline and budg-
Lab Servers use membrane etary demands—while ensuring research integrity.
technology to produce up Sorbtech delivers a wide range of solutions for prepar-
to 400 SLPM of high-pu- ative chromatography, processs manufacturing, flash
rity nitrogen from exter- chromatography, and purification. Our consultative in-
nal compressed air sources. dustry experts build long-term partnerships with the
Available in both wallmount- laboratories we serve and are ready to provide you with
able and floor-stand config- a broad offering of high performance, high purity prod-
urations, the generators are ucts. These include:
space-saving solutions that
maintain quiet and steady • Silica Gel
operation. Proton OnSite Ni- • Bonded Phases
trogen Lab Servers can re- • Alumina
place multiple benchtop • Polymeric Resins
generators and provide ex- • Size-Exclusion Media
cess capacity for addition-
al instrumentation. Lab Servers can be integrated into
Contact us today to see how we can improve your
an existing air system, or can be paired with a dedicat-
laboratory, pilot, and process applications. Check out
ed compressor system. Nitrogen Lab Servers are more
our current promotions today at www.sorbtech.com, or
cost effective to install and maintain compared to in-
call 770-936-0323.
stalling multiple benchtop gas generators.

Proton OnSite Sorbent Technologies

Wallingford, CT Norcross, GA
www.protononsite.com www.sorbtech.com

VOL 1 • NO 3 • SEPTEMBER/OCTOBER 2018 www.CannabisScienceTech.com | CANNABIS SCIENCE AND TECHNOLOGY

 CALL FOR PAPERS
Cannabis Science and Technology:
+BOVBSZ 201 Issue
Cannabis Science and Technology magazine is seeking contributed
manuscripts for the +BOVBSZ
BOE
.BSDI 2019 issues.
Manuscript scope and format
Cannabis Science and Technology broadly covers science and product quality issues in the cannabis industry.
We welcome manuscripts that describe analytical methods for product quality control (including sample preparation
techniques); the development of standard (consensus) methods; proper laboratory techniques and laboratory best
practices; laboratory accreditation, proficiency testing, and interlaboratory comparison testing; equipment
and technology for testing and processing; regulatory issues; current good manufacturing practices (cGMPs);
and research on cannabinoids and terpenes.

Technical articles
Technical articles describe improvements in methods or techniques. Papers should be ~3500-4500 words long and
should be of immediate relevance to the analytical and quality issues facing the cannabis industry. Authors should
not make comparisons between commercially available products from different manufacturers. We urge authors
to submit a proposal to the editor before completing a manuscript.
Tutorial articles
Tutorial articles should be presented as a how-to on a technique, application, or method related to cannabis analysis
or processing (
for example, QuEChERS extraction). Papers should be ~2000-2500 words long and should be of
immediate relevance to the analytical and quality issues facing the cannabis industry. We urge authors to submit
a proposal to the editor before completing a manuscript.
Review articles
Review articles survey recent developments and the state of the art of current techniques or emerging technologies.
Manuscripts should be ~3500-4500 words long. We urge authors to submit a proposal to the editor before completing
a manuscript.

Deadlines
Submission deadlines for the +BOVBSZ

issue are:
Abstracts: /PWFNCFS



Completed manuscripts: %FDFNCFS



Submission deadlines for the .BSDI 2019 issue are:

Abstracts:
+BOVBSZ
, 201

Completed manuscripts:


'FCSVBSZ



Where to submit
Send all proposals and completed articles to Editor Meg L’Heureux, at [email protected] (tel. +1.732.346.3051).
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There was
“There s an amazing energy level
vel a
at this year’s
year s show, a coming
together of an
analytical, medical and
a d cannabis experts that felt like
an extended cannabis family!”
- Tracy Ryan
n (CannaKids and SavingSophie.org
rg)

@CannabisScienceConference
CannabisScienceConference.com
@CannabisScienceConference
Sponsors
Sponsorship
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o are available.
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m forr more
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on
 Sample prep
made simple.
For LC, GC, MS,
& ICP analysis.

For over 40 years, CEM has been a trusted

source for analytical sample prep systems.

Our passion is to develop systems that are

simple to use, cost effective to operate, and
include industry leading technical support; for
those times when you need it.

EDGE™ MARS™ 6
Extraction system for potency, Microwave digestion system
pesticides, terpenes, & mycotoxins for heavy metals
cem.com/EDGE cem.com/MARS6

We Simplify Science