The Ever Changing Scope of Laboratory Testing

Laboratory testing is often thought of, in popular culture, as a simple and straightforward process. The popularity of crime dramas like CSI has proliferated the idea that sample testing simply involves adding a pinch of a sample to a machine and, with the press of a button, out pops an answer! We are sure many analysts wish it was just that easy!

While it is true that with the evolution of analytical instrumentation it is now possible to see a greater variety of compounds at very low concentrations, the reality of analytical testing and laboratory processes is often far from a one button answer. More often than not, laboratories are challenged with highly regulated and difficult sample preparation, extraction and testing procedures, covering a wide variety of sample types – ranging from agricultural to zoological specimens and beyond. And now, at SPEX, we count ourselves very lucky to have a front row seat to witness the birth, growth and regulation of a brand new industry around a brand new sample type.

By legalizing marijuana, recent legislation has opened up a new agricultural and testing industry around Cannabis. However, its federal status as a Class I drug has prevented federal regulating bodies from issuing recommendations and testing methods, leaving local testing laboratories at the forefront of testing regulation and legislation. The science and state regulatory communities are now working together to define the roles and targets for testing in this new industry, as well as, redefine global strategies for testing harmonization worldwide.

Below, we will take a look at a range of testing questions; from new analytical fields to well established industries which are being more tightly controlled and refined. This issue will examine new research being conducted in the analytical field, and take a fresh look at some well explored areas that are gaining new attention in the research world.

The Wild West of Analytical Testing

By Patricia Atkins, SPEX CertiPrep, Applications Scientist

The legalization of Cannabis, in certain states, has started a gold rush of growing facilities and testing labs, all trying to shape law and regulation out of what was previously an unregulated illegal product. Matthew Haskin, President and Co-Founder of CannaSafe Analytics, took some time out of his busy schedule to talk with SPEX CertiPrep about this growing industry and the challenges that are presenting themselves in this new analytical field.

CannaSafe Analytics started with a need for medical Cannabis users to know what they were consuming. Through family medical challenges, Matt was inspired to start a Cannabis testing laboratory dedicated to testing the potency and safety of medical Cannabis. Over the past six years, CannaSafe Analytics has grown and progressed to the forefront of Cannabis testing and regulations.

CannaSafe was the first Cannabis testing laboratory to be awarded an ISO 17025:2005 accreditation, setting a high standard for other Cannabis testing labs to follow. Matt is a member of the California Cannabis Association and NORML, a Cannabis law reform organization. In addition to being a member of the California Cannabis Association and NORML (a Cannabis law reform organization), Matt is a member and advisor to several regulatory boards looking to create the regulations for Cannabis testing and safety.

How did you get involved with Cannabis testing?

It started about six years ago, due to family medical issues. We turned to medical Cannabis and wanted to ensure its safety and efficacy. But, there was just nowhere to go. It got the wheels turning. I approached my father, who was an organic chemist, and we started developing the concept, building the team and assembling the players. It snowballed from there.

Who were the first customers who came to you for testing?

Our first customers came to our California laboratory, where we started. Our primary customers were 50% dispensaries and 50% growers; maybe not 50% because we did get actual patients who came in and wanted to know where their medicine falls in with all this, or the patient got it from another caregiver and they want to be sure it was safe. Those clients were about maybe 10%.

We work with a lot of growers and dispensaries that voluntarily want to regulate themselves and get testing. The bulk of what we do in California is potency analysis with some basic safety screening. We are still trying to get them in this self-regulatory mode to, at least, determine potency.

The last year, or maybe year and a half, we have seen a shift. We are starting to get more requests for contamination testing: certain microbial assays and more generic pesticide residue testing. The bulk of our testing is still potency analysis, meaning cannabinoids and terpenes.

What form are your samples?

We do all forms: topicals (we have seen a lot of topicals recently), tinctures, pure concentrates (meaning wax shatters), flowers, and edibles. Edibles are becoming bigger very quickly. Edibles are a huge group of samples that need to be labelled. The fastest growing segment is the edibles, then the concentrates.

When you started testing, what types of samples were you seeing in the lab?

At first, it was 60% flower, 20% edibles and 20% concentrates. Now it is at least 40-50% edibles, 30% concentrates and 30% flowers. Edibles and concentrates have come on really strong. We are doing a lot more residual solvent type testing for the concentrates.

What are the biggest changes you have seen from when you started to now?

We are seeing a new wave towards contamination testing; to really understand if we are harming ourselves in some manner that we are not aware of. It is all part of education. Being underground and black market for so long, the worry was not about it being harmful, just illegal. Now that legalization has happened, the question becomes: Is this safe for me? Consumers believe in the plant but don’t know what’s on the plant.

Contamination is the biggest change from the California perspective. The dispensaries are now starting to want their product checked for contamination; testing for groups of pesticides like carbamates, or for yeast and mold content. California is not yet at the same point as some other states, which are doing an entire panel from pesticide residues and all microbials, to heavy metals and aflatoxins. California dispensaries are starting to go down that route.

Are you seeing requests for terpene profiles, especially in the light of medical research regarding Cannabis and terpenes?

Terpenes are becoming very big due to this new wave of education. We are going to see patients getting away from wanting specific varieties and start to say they want something for sleep, depression, or something uplifting. We will be looking to terpene profiles to determine which strains will be recommended.

What are the three most important chemical analysis targets?

First is cannabinoid potency, you have to have the potency labelled. Pesticide residue will have to be number two, and number three we will call the overall microbial area, which consists of at least six different assays. Then, we can start getting into the other targets like metals and mycotoxins.

What are your sample preparation methods? Are they different for each type of product? What type of instrumentation are you using?

Depending on which assay we are looking at, the sample prep is different. The cannabinoid and terpene profiles are pretty simple shake and shoot methods. For pesticide residues, we’re doing more QuEChERS to eliminate some of that matrix interference. Method development is definitely still ongoing. I don’t think any lab is ready for over 400 pesticide analytes and all the interferences yet.

The cannabinoids and terpenes all resolve very well on both GC and LC combined with mass spec to be sure we are identifying the correct compounds. As the reference materials manufacturers, like SPEX CertiPrep, are providing more and more standards for the industry, it certainly helps with out analyses. We are growing in our instrumentation. We are trying to be allinclusive. We offer GC, HPLC, and LC-QQQ for pesticide residues, and ICP for metals.

Where do you think the industry is heading for sample prep and methodology? Do you think there will be standardized methods and SOP’s?

I think that is a ways off. I know with edibles, it is not a shake and shoot method. There is a lot of sample prep from solvent exchange, liquid-liquid extraction to many other types of preparation. We are trying all these different ways since the matrices are so complex: chocolates, ice cream, hard candy, and potato chips…it is all over the board. We are trying to determine what the best prep method is to recover what we are looking for. I think those methods will be kept proprietary by the labs as long as they can be. Each lab is working very hard to develop those methods and no one will be willing to hand over their hard work to other labs not working on methods right now. I don’t see the sample prep becoming part of a standardized method.

I do see the instrument methods becoming standardized. Right now we are working in Nevada to recommend instrument methods that we will ultimately require between all labs. Ten years from now, someone will license the method or methods will be published and adopted that everyone will end up following.

What methodologies and protocols are you using to do method development?

I would say EPA and USP have methods we use as templates to create our own in-house methods. We use FDA methods and AOAC methods for microbial analysis. Sometimes the methods for FDA and AOAC are not feasible. For example, one salmonella method requires preparation of 20-25 grams or more and prefers 100 grams of sample. No one wants to give us 100 grams to run one salmonella assay for them. It is too expensive. It also translates to the pesticide residue. Even the USP method for botanical products requires 10 grams. If I tell one of my clients I need 10 grams for one pesticide residue, they are not happy. They will go to the next lab which says they only need 2 grams.

Because we are the laboratory, and our customers are the producers and growers, if we come back with unsafe levels of contamination and those batches fail, the clients are not happy. There are laboratories out there doing bad science. Those labs cannot prove they can detect an analyte or have detection limits insufficient to the analysis they are doing. Some potentially unsafe batches could be passing in those labs and the labs are being rewarded for bad science. Producers flock to them. Guess where the clients are going? Sometimes it feels as if we are being punished for good science.

Where do you think the regulations and testing are heading?

I think a lot of the states will continue down the same path of more and more requirements for testing. Massachusetts, Connecticut and now Nevada are coming out with very strict testing requirements. I see other states adopting those standards and adopting more.

What do you think the timeline is for Cannabis testing? I know you have been working with Nevada and California.

Nevada has regulations in place and we are now fine tuning what was left out of the regulations. For example, the regulations require pesticide residue analysis, but what does that mean? We are trying to define what that means. Nevada regulations have already said requirements will include: pesticide residue, a panel of six different microbial assays, mycotoxins, and metals. We have to define the limits for all of that. In Nevada, the regulations are written and we are fine tuning them. In states like Washington, they have some ways to go yet. For example, Washington states that labs have to do microbial and potency testing and there must be spot-checking for pesticide residues and heavy metals. That was their approach, but to date, I do not think the spot-checking has occurred yet. In California, I think we will go legal this year and it will take another year or so to write and implement regulations similar to Nevada. Then, there is the new twist of the tribal nations and what they might be able to do now and in the future with Cannabis. Those rulings will be gamechangers. (Note: At the end of 2014, the US Justice Department gave the tribal nations the authority to authorize the legalization of Cannabis on tribal nation lands and told federal prosecutors that any issues would be addressed on a “government-togovernment basis”.)

What are the challenges in Cannabis testing?

We are always challenged by sample prep for edibles. We keep moving into more indepth testing like metals or multi-residue pesticide. These are the new challenges we are starting to see.

We can mimic some of the methods for some of the other agriculture products for pesticide residues, but they have to be validated across the Cannabis matrix. When regulations do hit, like in Nevada, then what does that mean? There are no methods for that, how do we develop that? How do laboratories all stay on the same playing field?

Another challenge we have are sample limitations and sampling requirements to do good science. The cannabinoids and terpene analyses actually use only little material. The biggest challenge is having enough material to feel like you have a representative homogeneous sample.

What myths and misconceptions do you find are prevalent in the Cannabis testing community about Cannabis testing?

Standard deviations! Standard deviations between laboratories or amongst the industry.

We, and fourteen other laboratories, did our first proficiency testing ever in a third party PT program with Emerald Scientific last fall. The reported numbers were all over the board to the actual concentrations. Our labs did very well, but, there were results from other labs that were up to 30% over and 30% under the known values. That is kind of alarming.

We see that misinformation especially when translating into clients testing edibles. Recovery levels and spiking are not clearly understood by laboratories and their clients. Some laboratories do not comprehend the need and procedures for recovery studies. There is also a problem with sampling (i.e. about what parts of the sample and how much you pull). What part of the flower did you pull from? We use very little flower, about 100 mg. No matter how homogenous you feel you get that sample, it is still going to play on what you pulled with such a small sample size. It will create variations. This point of needing more recovery and accuracy data is the educational point we are trying to get across to the industry.

How do you think laboratories improve homogeneity and reduce error and variation?

Most labs are trying to homogenizer the best they can. Cryogrinding is new to our lab. We just got your SPEX SamplePrep GenoGrinder. I think that is the next step for laboratories to ensure pure homogenization of the products. In a short time we have gone from the absolute gunslinger testing, to where it is finally getting to the point there is some oversight and regulations. A lot of what we are hearing now is a lot of labs are moving towards ISO 17025:2005 accreditation. A lot of laboratories are going to work on getting accreditation. I think it will go a long way to weed out the bad science.

Matthew Haskin is the co-founder and president of CannaSafe Analytics in California. He also functions as their Laboratory Manager. He holds a BS from Columbia State University and has experience in domestic and international business. CannaSafe Analytics is a full service Cannabis testing laboratory dedicated to addressing the concerns over Cannabis safety and became the first Cannabis laboratory to obtain ISO 17025:2005 accreditation.

The Wild West of Analytical Testing

Terpenes are a large, varied class of strongsmelling compounds usually associated with plants and other botanicals. Terpenes are hydrocarbons, while their associated terpenoids (also known as isoprenoids) are terpenes that have some rearrangement or oxidation to create assorted functional groups such as alcohols, esters, etc.

Terpenes are the major component of resins and turpentine. The term terpene is derived from the compound turpentine. Terpenes and terpenoids are the primary compounds in many types of botanical and essential oils. These oils are used as natural flavor and fragrance sources and are often used in the perfume industry. Historically, terpenes, through essential oils and botanicals, have been used in holistic practices and aromatherapy.

In nature, terpenes are found in many common plants, including coniferous trees. These terpenes and terpenoids have natural pesticide properties and are thought to be part of the plant’s natural defense system. These terpene compounds are often offgassed by plants and trees in warm weather and are responsible for the characteristic blue haze of the Smoky Mountains and the Blue Ridge Mountains. This cloud of terpenes can act as a natural form of air pollution.

Many consumer products, including perfume, flavorants, wine and beer, are dependent upon terpenes for the character, flavor and fragrances they impart upon the products. The flavor and aroma of hops are critical to beer, especially Myrcene, beta-Pinene, beta-Caryophyllene, and alpha-Humulene. The floral notes of wine grapes can be traced back to terpenoid compounds such as Damascene and Geraniol, which are also found in roses. The diversity of terpenes and terpenoids are recognized by all the ranges of scents and flavors they produce; from the pine scent of Pinene (the most widely encountered terpene in nature) to the lavender and mint notes associated with Linalool. A good resource in looking at more terpenes is the essential oil database. http://nipgr.res.in/Essoildb/index.html

The newest botanical product to be profiled for terpenes is Cannabis. Since Cannabis and beer hops are both part of the Cannabacea family, it is not hard to see why terpene analysis of Cannabis would be of interest.

Cannabis growers and seed companies are becoming increasingly interested in creating terpene profiles for their products. The Green House Seed Company was one of the first companies to post terpene profiles of their seed products. Matt Haskin, president of CannaSafe Analytics, a Cannabis testing laboratory, sees the demand for terpene testing growing at a rapid pace. (See “The Wild West of Analytical Testing”).

Terpene profiles are used as a way to characterize and fingerprint different varieties of Cannabis for specific therapeutic uses, appropriate for a range of medical conditions. Terpenes such as beta-Myrcene and Nerolidol have sedative properties. Limonene has anti-inflammatory properties.

Studies show that the terpenes in Cannabis work synergistically with the cannabinoid compounds to enhance the therapeutic effect. http://onlinelibrary.wiley.com/enhanced/doi/10.1111/j.1476-5381.2011.01238.x/

The extraction and analysis of terpenes in the analytical laboratory can be challenging, especially when confronted with sample preparation, sample clean-up and dealing with matrix effects. In many cases, related terpenes have the same or similar mass spectral fingerprints by GC/MS, making them difficult to identify in complex mixtures where many isomers or similar compounds are present. In additional to difficult analyses, there has been a lack of suitable reference materials and standards.

In late 2014, SPEX CertiPrep was proud to be one of the first standard companies to produce terpene standards to aid the analytical community with their terpene profile analyses. Our terpene standard mixes allowed the analyst to identify and quantitate a wide range of common terpenes found in hops and Cannabis.

Regulating the Regulations

FDA (US Food & Drug Administration)

FDA: www.fda.gov

FDA Newsroom: http://www.fda.gov/NewsEvents/Newsroom/default.htm

Recalls, Market Withdrawals, & Safety Alerts: http://www.fda.gov/Safety/Recalls/default.htm

EPA (US Environmental Protection Agency)

EPA: www.epa.gov

EPA Newsroom: https://www.epa.gov/newsroom

Analytical Methods and Procedures for Pesticides https://www.epa.gov/pesticide-analytical-methods

USP (US Pharmacopeial Convention)

USP: www.usp.org

USP Newsroom: http://www.usp.org/news

Compliance with USP’s Standards for Herbal Supplements http://qualitymatters.usp.org/what-it-means-be-compliance-usp-sstandards-herbal-supplements

ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use)

ICH: www.ich.org

ICH Newsroom: http://www.ich.org/ichnews/newsroom.html

REACH (International Uniform Chemical Information Database)

REACH: http://iuclid.eu

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