Tag Archives: laboratories

ASTM International Begins Crafting Cannabis Standards

By Aaron G. Biros
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Last week, the American Society for Testing and Materials (ASTM International) approved the formation of a committee to develop standards for the cannabis industry. ASTM International is a standards development organization that develops voluntary consensus-based standards for industries. United States regulatory bodies and the World Trade Organization have recognized the organization’s standards in other industries.

On March 1st, the non-profit announced the formation of a committee for ““creating technical standards and guidance materials for cannabis and its products and processes.” So now that the vote has passed, what is the next step? They will begin the process of member training, appointment of leadership and writing the bylaws. ASTM will have two online briefings before their official meeting for the cannabis committee (D37) in June. Those meetings will discuss how the committee was formed and how it’ll be structured. The first official meeting of the cannabis committee will take place June 11th and 12th in Toronto.

Lezli Engelking
Lezli Engelking, founder of FOCUS

Voluntary consensus-based standards means there is a balance of interests, an appeals process and an overall consensus has been reached. The areas of focus for the cannabis standards include indoor and outdoor horticulture and agriculture, quality management systems, laboratories, processing and handling, security and transportation, and personnel training, assessment and credentialing. Many standards will be developed under each of these broad categories. A large component of consensus-based standard development is openness…so anyone who wants to participate in the development of the standards is welcome and encouraged to do so. They are still looking for participants from the cannabis industry and those interested can register here.

Lezli Engelking, founder of the Foundation of Cannabis Unified Standards (FOCUS), says this is terrific news for the cannabis industry. “To have a global organization like ASTM, that federal governments actually work with and respect, is a huge stride forward for the cannabis industry,” says Engelking. “FOCUS is thrilled to be working with ASTM.” FOCUS and ASTM International have a derivative work license agreement that provides ASTM the FOCUS standards to use as a baseline for developing their standards. “FOCUS will continue to certify cannabis businesses to the FOCUS standards, but we will be able to add in the ASTM standards to our certification platform,” says Engelking. “It helps us expand our depth and reach in tools for our clients.”

FOCUS standards and ASTM standards are both voluntary consensus-based, meaning it is the businesses and stakeholders participating that ultimately write the standards. The organizations’ staff does not actually contribute to and develop the standards; they are more like a vehicle for the industry and stakeholders to come to a consensus, according to Engelking. “ASTM does the same thing that we do for the cannabis industry, just on a much larger scale,” says Engelking. “Its role is to fulfill the development, not actually develop it.” Because of that, ASTM and FOCUS standards can work in harmony.

OLCC-Logo

OLCC Issues First Recreational Cannabis Recall for Oregon

By Aaron G. Biros
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OLCC-Logo

On March 18th, the Oregon Liquor Control Commission (OLCC) issued its first recall for recreational cannabis products. The recall, according to the press release, occurred because an unnamed wholesaler sent cannabis products to a retailer before the pesticide test results were entered into the OLCC Cannabis Tracking System (CTS).


Photo: Michelle Tribe, Flickr

The cannabis grown at Emerald Wave Estate, LLC is said to fail a test for pyrethrins exceeding the Oregon Health Authority (OHA) action level (the action level for pyrethrins is 1 ppm). Pyrethrins are a class of insecticides derived from the chrysanthemum flower. Their toxicity varies a lot depending on exactly what organic compound was used, but has an acute toxicity level that is cause for concern. When exposed to high levels of pyrethrins, people have reported symptoms similar to asthma. Generally, pyrethrins have a low chronic toxicity for humans.

The retailer, Buds 4 U LLC, located in Mapleton, OR, issued a voluntary recall for 82.5 grams of the strain Blue Magoo sold between March 8th and 10th. After finding the failed test results in the CTS, the retailer immediately contacted the OLCC. According to The Portland Tribune, OLCC spokesman Mark Pettinger says the retailer was very cooperative in immediately notifying the OLCC. “The retailer was great,” says Pettinger. “They get the gold star.” The Portland Tribune also says the wholesaler who shipped the cannabis prior to test results being entered is Cascade Cannabis Distributing of Eugene. That mistake could be a violation of Oregon’s regulations, leading to a 10-day closure and up to a $1,650 fine.

According to the press release, the rest of the nine pounds in the batch is on hold “pending the outcome of an additional pesticide retest.” The OLCC encourages consumers to check if their products have the license and product numbers detailed in the press release. They advise consumers who did purchase the affected cannabis to dispose of the product or return it to the retailer. The press release also mentions that they have not received any reports of illness related to the tainted cannabis.

Cannabis-Specific Certified Reference Materials

By Aaron G. Biros, Don Shelly
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A certified reference material (CRM) is generally recognized as providing the highest level of traceability and accuracy to a measurement. A CRM designed specifically for cannabis testing and tailored to state-specific testing regulations could help laboratories better ensure the safety of their products.

The fact that a certificate accompanies a reference material does not qualify it as a CRM. The reference material must be produced in accordance with ISO Guide 34 specifications by an accredited manufacturer. Adam Ross, key account manager and organic specialist at LGC Standards, says accreditation is a big part of bringing legitimacy to cannabis testing. “For a laboratory to receive an ISO 17025 accreditation, they must purchase their RMs from an ISO 17025 manufacturer. The best option is to purchase an ISO Guide 34 manufactured CRM,” says Ross. “It is particularly important for testing requirements, such as potency, pesticides, etc., where quantitation is expected, to use properly certified quantitative reference materials.” LGC Standards, a 175-year-old company, is one of those manufacturers that invested the time and money to achieve ISO Guide 34 accreditation and offers a spectrum of CRMs for cannabis testing.

Adam Ross, LGC Standards
Adam Ross, LGC Standards

The major advantage to using a proper CRM is an increased level of credibility. Auditors recognize the value of using a CRM which can add to the integrity of the results produced. The regular use of certified reference standards along with proper training, methodology and instrumentation, will facilitate a result that has the least amount of uncertainty and is more defendable. “The regular use of certified reference standards will help ensure products that go to market are safe to consume,” says Ross.

With regard to potency analyses, Ross has some key insights to help a laboratory better utilize CRMs. “My advice? Don’t mix the cannabinoids; labs analyzing by GC/FID have discovered that some of the cannabinoids will co-elute. Also, they have a short shelf life when mixed together,” says Ross. “Cannabinoid analysts should use GC/MS or LC/MS for their analysis or analyze the cannabinoids individually,” says Ross.

rsz_cannabis_product_photo_lgc-1So what happens if a cannabis lab uses non-certified reference materials? Labs might save money in the short term. CRMs are slightly more expensive than a non-certified reference material, but will increase the defensibility of a lab’s data. Using a reference material created in-house or from a non-accredited vendor can lead to less-than-accurate results. A non-certified reference material has a greater chance of being made incorrectly. The publication of incorrect data damages the credibility of the testing lab and could lead to legal action against the lab from damaged parties.

One of the major challenges for the cannabis testing industry is the variation in state-to-state regulations. Ross says that Oregon’s regulations are pretty comprehensive and that other states should look to the Oregon Environmental Laboratory Accreditation Program (ORELAP) for guidance. According to Ross, ORELAP would like to see higher quality standards with legitimate traceability. Utilizing CRMs the correct way will help laboratories achieve greater accuracy.

Here are some tips for using CRMs appropriately:

  • Always bring your standards to room temperature before making a dilution.
  • Matrix matched calibration standards provide more accurate quantitation. Prepare standards in the solvent from extracted blank matrices.
  • Always bracket your analytical runs with continuing calibration verification standards. Proving that your instrument remained calibrated during the run gives your data more credibility.

Analytical chemists purchase CRMs for three primary uses in the testing lab:

  • To calibrate the instrument that will be used to perform the testing
  • To confirm the instruments continuing calibration throughout the analytical process
  • For analytical quality control or “spikes”

Typically, labs will spike known concentrations of the analytes of interest into a control sample and regular samples with the intent of testing analytical efficiency. Recoveries of analytes from the spiked control sample tell the chemist how well the analytical method is working. The spiked samples (matrix spikes) demonstrate to what extent the sample matrix (the consumable being tested) is influencing the results of the analytical procedure.

CRMs could be described as the nexus between cannabis testing results, the human element and the instrumentation used in an analysis. By using a cannabis-specific CRM, the cannabis testing community can demonstrate tangible improvements in accuracy and legitimacy.

From The Lab

QuEChERS 101

By Danielle Mackowsky
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Sample preparation experts and analytical chemists are quick to suggest QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) to cannabis laboratories that are analyzing both flower and edible material for pesticides, mycotoxins and cannabinoid content. Besides having a quirky name, just what makes QuEChERS a good extraction technique for the complicated matrices of cannabis products? By understanding the chemistry behind the extraction and the methodology’s history, cannabis laboratories can better implement the technology and educate their workforce.

QuEChERS salt blends can be packed into mylar pouches for use with any type of centrifuge tubes
QuEChERS salt blends can be packed into mylar pouches for use with any type of centrifuge tubes

In 2003, a time when only eight states had legalized the use of medical cannabis, a group of four researchers published an article in the Journal of AOAC International that made quite the impact in the residue monitoring industry. Titled Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and “Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce, Drs. Michael Anastassiades, Steven Lehotay, Darinka Štajnbaher and Frank Schenck demonstrate how hundreds of pesticides could be extracted from a variety of produce samples through the use of two sequential steps: an initial phase partitioning followed by an additional matrix clean up. In the paper’s conclusion, the term QuEChERS was officially coined. In the fourteen years that have followed, this article has been cited over 2800 times. Subsequent research publications have demonstrated its use in matrices beyond food products such as biological fluids, soil and dietary supplements for a plethora of analytes including phthalates, pharmaceutical compounds and most recently cannabis.

QuEChERS salts can come prepacked into centrifuge tubes
QuEChERS salts can come prepacked into centrifuge tubes

The original QuEChERS extraction method utilized a salt blend of 4 g of magnesium sulfate and 1 g of sodium chloride. A starting sample volume of 10 g and 10 mL of acetonitrile (ACN) were combined with the above-mentioned salt blend in a centrifuge tube. The second step, dispersive solid phase extraction (dSPE) cleanup, included 150 mg of magnesium sulfate and 25 mg of primary secondary amine (PSA). Subsequent extraction techniques, now known as AOAC and European QuEChERS, suggested the use of buffered salts in order to protect any base sensitive analytes that may be critical to one’s analysis. Though the pH of the extraction solvent may differ, all three methods agree that ACN should be used as the starting organic phase. ACN is capable of extracting the broadest range of analytes and is compatible with both LC-MS/MS and GC-MS systems. While ethyl acetate has also been suggested as a starting solvent, it is incompatible with LC-MS/MS and extracts a larger amount of undesirable matrix components in the final aliquot.

All laboratories, including cannabis and food safety settings, are constantly looking for ways to decrease their overhead costs, batch out the most samples possible per day, and keep their employees trained and safe. It is not a stretch to say that QuEChERS revolutionized the analytical industry and made the above goals tangible achievements. In the original publication, Anastassiades et al. established that recoveries of over 85% for pesticides residues were possible at a cost as low as $1 per ten grams of sample. Within forty minutes, up to twelve samples were fully extracted and ready to be analyzed by GC-MS, without the purchase of any specialized equipment. Most importantly, no halogenated solvents were necessary, making this an environmentally conscious concept. Due to the nature of the cannabis industry, laboratories in this field are able to decrease overall solvent usage by a greater amount than what was demonstrated in 2003. The recommended starting sample for cannabis laboratories is only one gram of flower, or a tenth of the starting volume that is commonly utilized in the food safety industry. This reduction in sample volume then leads to a reduction in acetonitrile usage and thus QuEChERS is a very green extraction methodology.

The complexity of the cannabis matrix can cause great extraction difficulties if proper techniques are not used
The complexity of the cannabis matrix can cause great extraction difficulties if proper techniques are not used

As with any analytical method, QuEChERS is not perfect or ideal for every laboratory setting. Challenges remain in the cannabis industry where the polarity of individual pesticides monitored in some states precludes them from being amenable to the QuEChERS approach. For cannabis laboratories looking to improve their pesticide recoveries, decrease their solvent usage and not invest their resources into additional bench top equipment, QuEChERS is an excellent technique to adopt. The commercialization of salt blends specific for cannabis flowers and edibles takes the guesswork out of which products to use. The growth of cannabis technical groups within established analytical organizations has allowed for better communication among scientists when it comes to best practices for this complicated matrix. Overall, it is definitely worth implementing the QuEChERS technique in one’s cannabis laboratory in order to streamline productivity without sacrificing your results.

The Practical Chemist

Pesticide Analysis in Cannabis and Related Products: Part 3

By Julie Kowalski
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As mentioned in Part 1, pesticides residue analysis is very challenging especially considering the complexity of cannabis and the variety of flower, concentrates and infused products. In addition, pesticides are tested at low levels typically at parts-per-billion (ppb). For example, the food safety industry often uses 10 ppb as a benchmark limit of quantification. To put that in perspective, current pesticides limits in cannabis range from 10 ppb default (Massachusetts Regulatory Limit) to a more typical range of 100 ppb to 2 ppm in other states. Current testing is also complicated by evolving regulations.

Despite these challenges, adaptation of methods used by the food safety industry have proved successful for testing pesticides in cannabis. These methods typically rely on mass spectrometric detection paired with sample preparation methods to render the sample clean enough to yield quality data.

Pesticide Analysis Methods: Sample preparation and Analytical Technique Strategy

Generally, methods can be divided into two parts; sample preparation and analytical testing where both are critical to the success of pesticide residue testing and are inextricably linked. Reliance on mass spectrometric techniques like tandem mass spectrometry and high resolution accurate mass (HRAM) mass spectrometry is attributed to the substantial sensitivity and selectivity provided. The sensitivity and selectivity achievable by the detector largely dictates the sample preparation that will be required. The more sensitive and selective the detector, the less rigorous and resource intensive sample preparation can be.

Analytical technique: Gas and Liquid Chromatography Tandem Mass Spectrometry 

The workhorse approach for pesticide residue analysis involves using gas chromatography and liquid chromatography tandem mass spectrometry (MS/MS) in the ion transition mode. This ion transition mode, often referred to as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM), adds the selectivity and sensitivity needed for trace level analysis. Essentially, a pesticide precursor ion is fragmented into product ions. The detector monitors the signal for a specified product ion known to have originated from the pesticide precursor ion. This allows the signal to be corrected, associated with the analyte and not with other matrix components in the sample. In addition, because only ions meeting the precursor/product ion requirements are passed to the detector with little noise, there is a benefit to the observed signal to noise ratio allowing better sensitivity than in other modes. Even though ion transitions are specific, there is the possibility a matrix interference that also demonstrates that same ion transition could result in a false positive. Multiple ion transitions for each analyte are monitored to determine an ion ratio. The ion ratio should remain consistent for a specific analyte and is used to add confidence to analyte identification.

The best choice for pesticide analysis between gas chromatography (GC) and liquid chromatography (LC) is often questioned. To perform comprehensive pesticide screening similar to the way the food safety market approaches this challenge requires both techniques. It is not uncommon for screening methods to test for several hundred pesticides that vary in physiochemical properties. It may be possible that with a smaller list of analytes, only one technique will be needed but often in order to reach the low limits for pesticide residues both GC and LC are required.

Modified QuEChERS extraction using 1.5 grams of cannabis flower. Courtesy of Julie Kowalski (Restek Corporation), Jeff Dahl (Shimadzu Scientific Instruments) and Derek Laine (Trace Analytics).
Modified QuEChERS extraction using 1.5 grams of cannabis flower. Courtesy of Julie Kowalski (Restek Corporation), Jeff Dahl (Shimadzu Scientific Instruments) and Derek Laine (Trace Analytics).

Analytical technique: Sample Preparation

Less extensive sample preparation is possible when combined with sensitive and selective detectors like MS/MS. One popular method is the QuEChERS approach. QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged and Safe. It consists of a solvent extraction/salting out step followed by a cleanup using dispersive solid phase extraction. Originally designed for fruit and vegetable pesticide testing, QuEChERS has been modified and used for many other commodity types including cannabis. Although QuEChERS is a viable method, sometimes more cleanup is needed and this can be done with cartridge solid phase extraction. This cleanup functions differently and is more labor intensive, but results in a cleaner extract. A cleaner extract helps to secure quality data and is sometimes needed for difficult analyses.

Colorado Cannabis Lab Methods Updated for Microbial Testing

By Aaron G. Biros
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The Colorado Department of Public Health and Environment’s (CDPHE) Marijuana Laboratory Inspection Program issued a bulletin on January 30th regarding updates required for licensed cannabis testing labs. The updated method for microbial contaminant testing includes a longer incubation period in yeast and mold testing.BannerForEnf

“After careful consideration of emerging data regarding the use and effectiveness of 3M Total Yeast and Mold Rapid Petrifilms in marijuana, CDPHE has concluded that 48 hours is not a sufficient incubation period to obtain accurate results,” the letter states. “Based upon the review of this information, marijuana/marijuana products require 60-72 hours of incubation as per the manufacturer’s product instructions for human food products, animal feed and environmental products.” The letter says they determined it was necessary to increase the incubation period based on data submitted from several labs, along with a paper found in the Journal of Food Protection.

An incubator (Right) at TEQ Analytical Labs
An incubator (Right) at TEQ Analytical Labs

According to Alexandra Tudor, manager of the microbiology department at TEQ Analytical Labs (a cannabis testing lab in Aurora, CO), the update is absolutely necessary. “The incubation time extension requirement from CDPHE offers more reliable and robust data to clients by ruling out the possibility of a false yeast and mold result during analysis,” says Tudor.

Alexandra Tudor, microbiology department manager at TEQ Analytical Labs
Alexandra Tudor, microbiology department manager at TEQ Analytical Labs

“3M, the maker of Petrifilm, recommends an incubation time of 48-72 hours, but during TEQ’s method validation procedure, we learned that 48-hour incubation was not sufficient time to ensure accurate results. Although some laboratories in industry had been incubating for the minimum amount of time recommended by the manufacturer, the 48-hour incubation time does not provide a long enough window to ensure accurate detection of microbiological contaminants present in the sample.” Tudor says the update will help labs provide more confident results to clients, promoting public health sand safety.IMG_6386-2

As a result of the update in testing methodology, cultivators and infused product manufacturers in Colorado need to submit a batch test for yeast and mold. The point of requiring this batch test is to determine if the producer’s process validation is still effective, given the new yeast and mold testing method.

emerald test retail

Analyzing The Emerald Test Results: Cannabis Labs Making Progress

By Aaron G. Biros
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emerald test retail

The Emerald Test advisory panel recently convened to review the results from the Fall 2016 round of the semi-annual Inter-Laboratory Comparison and Proficiency Test (ILC/PT), ahead of the third annual Emerald Conference just a few weeks away. After reviewing and analyzing the results, the panel noticed a significant improvement across the board over their Spring 2016 round of proficiency testing.rsz_emerald-scientific_letterhead-1

Emerald Scientific’s ILC/PT program is a tool laboratories use to check how accurate their testing capabilities are compared to other labs. A lab receiving The Emerald Test badge indicates their testing meets the criteria established by the panel to demonstrate competency. This means that they were within two standard deviations of the consensus mean for all analytes tested, according to Wes Burk, vice president of Emerald Scientific. He says the labs performed better than expected on both the microbial and pesticide tests.

Wes Burk, vice president of Emerald Scientific.
Wes Burk, vice president of Emerald Scientific.

emerald test retailEach lab has access to raw, anonymized data including a consensus mean, z-scores and kernel density plots. This round measured how well 35 cannabis labs perform in testing for potency, pesticides, residual solvents and microbial contaminants such as E. coli, Salmonella, Coliform, yeast and mold.

The advisory panel includes: Robert Martin, Ph.D., founder of CW Analytical, Cynthia Ludwig, director of technical services at AOCS, Rodger Voelker, Ph.D., lab director, OG Analytical, Tammie Mussitsch, QA manager at RJ Lee Group, Shawn Kassner, senior scientist at Neptune & Company, Inc., Jim Roe, scientific director at Steep Hill Labs, Chris Hudalla, Ph.D., founder and chief scientific officer at ProVerde Labs, Sytze Elzinga, The Werc Shop and Amanda Rigdon, Chief Technical Officer at Emerald Scientific.

amandarigdon
Amanda Rigdon, chief technical officer at Emerald Scientific

According to Amanda Rigdon, chief technical officer at Emerald Scientific, the labs performed very well in potency, residual solvents and microbial testing PTs. This is the first year the proficiency testing includes pesticides. “All of the labs did a great job identifying every pesticide in our hemp-based PT, but some more work will most likely have to be done to bring quantitative results in line,” says Rigdon. “Since this was the first pesticide PT we had offered, we were pretty conservative when choosing analytes and their levels. For the most part, analytes and levels were taken from the Oregon pesticide list, which is widely recognized to be the most reasonable and applicable pesticide list out there to date.” They covered pesticides of high concern, like abamectin and Myclobutanil, but also included a wide range of other pesticides that labs are expected to encounter.

Shawn Kassner, senior scientist at Neptune
Shawn Kassner, senior scientist at Neptune & Company, Inc.

Shawn Kassner, senior scientist at Neptune & Company, Inc., believes microbial contamination proficiency testing should be a priority for improving public health and safety going forward. Although five participating labs did not receive badges for the microbial contamination PTs, panel members say the overall performance was really quite good. “Microbiology testing are essential analyses for all cannabis products and it’s just slower in regulatory implementation than potency testing,” says Kassner. “The risk of Salmonella and E. coli to an individual using a medical cannabis product could be very life threatening. Microbiology contamination is a huge concern for any public health agency, which is why we have seen that microbiology testing is usually the first analytical test required after potency.” Kassner notes that there were few outliers and with each Emerald PT program, he is seeing an improvement in overall laboratory performance.

For The Emerald Test’s next round, the panel hopes to make some improvements in the test’s robustness and consistency, like obtaining assigned values for all samples and comparing to a consensus mean. “We want to develop permanent badge criteria, streamline the appeals process and possibly implement a qualitative performance review in the pesticide PT,” says Burk. For the next round of pesticide PTs, they want to build a better list of pesticides to cover more states, allowing labs to pick a set based on their state’s regulations. Burk says they also want to collect data on whether or not matrix-matched curves were used for pesticides.

Rodger Voelker, Cynthia Ludwig and Shawn Kassner, all members of the advisory panel, will be speaking at the Emerald Conference, discussing some of their findings from this round of proficiency testing. The Emerald Conference will take place February 2nd and 3rd in San Diego, CA.

The Practical Chemist

Potency Analysis of Cannabis and Derivative Products: Part 2

By Rebecca Stevens
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As mentioned in Part 1, the physiological effects of cannabis are mediated by a group of structurally related organic compounds known as cannabinoids. The cannabinoids are biosynthetically produced by a growing cannabis plant and Figure 1 details the biosynthetic pathways leading to some of the most important cannabinoids in plant material.

Potency figure 1
Figure 1: The biosynthetic pathway of phytocannabinoid production in cannabis has been deeply studied through isotopic labeling experiments

The analytical measurement of cannabinoids is important to ensure the safety and quality of cannabis as well as its extracts and edible formulations. Total cannabinoid levels can vary significantly between different cultivars and batches, from about 5% up to 20% or more by dry weight. Information on cannabinoid profiles can be used to tailor cultivars for specific effects and allows end users to select an appropriate dose.

Routine Analysis vs. Cannabinomics 

Several structurally analogous groups of cannabinoids exist. In total, structures have been assigned for more than 70 unique phytocannabinoids as of 2005 and the burgeoning field of cannabinomics seeks to comprehensively measure these compounds.¹

Considering practical potency analysis, the vast majority of cannabinoid content is accounted for by 10-12 compounds. These include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), Δ9-tetrahydrocannabivarian (THCV), cannabidivarin (CBDV) and their respective carboxylic acid forms. The cannabinoids occur primarily as carboxylic acids in plant material. Decarboxylation occurs when heat is applied through smoking, vaporization or cooking thereby producing neutral cannabinoids which are more physiologically active.

Potency Analysis by HPLC and GC

Currently, HPLC and GC are the two most commonly used techniques for potency analysis. In the case of GC, the heat used to vaporize the injected sample causes decarboxylation of the native cannabinoid acids. Derivatization of the acids may help reduce decarboxylation but overall this adds another layer of complexity to the analysis² ³. HPLC is the method of choice for direct analysis of cannabinoid profiles and this technique will be discussed further.

A sample preparation method consisting of grinding/homogenization and alcohol extraction is commonly used for cannabis flower and extracts. It has been shown to provide good recovery and precision² ³. An aliquot of the resulting extract can then be diluted with an HPLC compatible solvent such as 25% water / 75% acetonitrile with 0.1% formic acid. The cannabinoids are not particularly water soluble and can precipitate if the aqueous percentage is too high.

To avoid peak distortion and shifting retention times the diluent and initial mobile phase composition should be reasonably well matched. Another approach is to make a smaller injection (1-2 µL) of a more dissimilar solvent. The addition of formic acid or ammonium formate buffer acidifies the mobile phase and keeps the cannabinoid acids protonated.

The protonated acids are neutral and thus well retained on a C18 type column, even at higher (~50% or greater) concentrations of organic solvent² ³.

Detection is most often done using UV absorbance. Two main types of UV detectors are available for HPLC, single wavelength and diode array. A diode array detector (DAD) measures absorbance across a range of wavelengths producing a spectrum at each point in a chromatogram while single wavelength detectors only monitor absorbance at a single user selected wavelength. The DAD is more expensive, but very useful for detecting coelutions and interferences.

References

  1. Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids. Life Sciences, 78, (2005), pp. 539
  2. Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International, 98, (2015), pp. 1503
  3. Innovative Development and Validation of an HPLC/DAD Method for the Qualitative and Quantitative Determination of Major Cannabinoids in Cannabis Plant Material. Journal of Chromatography B, 877, (2009), pp. 4115

Rebecca is an Applications Scientist at Restek Corporation and is eager to field any questions or comments on cannabis analysis, she can be reached by e-mail, rebecca.stevens@restek.com or by phone at 814-353-1300 (ext. 2154)

First Nevada Cannabis Lab Receives ISO 17025 Accreditation.

By Aaron G. Biros
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On December 7th, 374 Labs received ISO 17025 accreditation, becoming the first in Nevada to do so. The laboratory, based in Sparks, Nevada, is state-certified and now the only ISO 17025 accredited lab in the state, according to a press release. The laboratory is a member in both the Association of Commercial Cannabis Laboratories (ACCL) and the Nevada Cannabis Laboratory Association (NVCLA).

Managing Partner Alec Garcia at the LCMS/MS
Managing Partner Alec Garcia at the LCMS/MS

“As Nevada transitions into an adult-use cannabis market, it’s very important that the state’s cannabis testing laboratories are held to the highest standards – and ISO 17025 is a requirement of top testing laboratories in all industries from biotech to forensics in most major countries,” says Dr. Jeff Angermann, assistant professor in the University of Nevada, Reno’s School of Community Health Sciences.

Laboratory Technician Bevan Meade working on sample preparation.
Laboratory Technician Bevan Meade working on sample preparation.

According to the release, 374 Labs was a driving force behind Nevada’s round robin cannabis lab testing program. That program, administered by the Nevada Division of Public and Behavioral Health (DPBH) and the Nevada Department of Agriculture (NDA), sends cannabis samples to each state-certified cannabis lab for a full analysis, measuring the consistency in test results across labs. “In other states proficiency involves testing pre-prepared, purified samples and neglects the challenges of coaxing out delicate analytes from the complex array of compounds found in actual marijuana,” says Laboratory Director Jason Strull. “I commend the DPBH and NDA for facilitating such an advanced quality program.”

Also notable is the announcing of their partnership with Clean Green Certified, a third-party certification (based on USDA organic certification) for sustainable, organically based cannabis cultivation. “Nevada allows certain levels of pesticides like Myclobutanil on its certified marijuana, so we wanted a way for patients and consumers to able to distinguish marijuana that is grown using organic methods,” said Laboratory Director Jason Strull. According to Michael Seibert, managing member of 374 Labs, they have already started performing inspections for the third-party certification and the first facility inspected was Silver State Trading in Sparks, Nevada (certified for both production and cultivation).

The C4 Cannabinomics Collaborative: Q&A with Dr. Zacariah Hildenbrand

By Aaron G. Biros
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Dr. Zacariah Hildenbrand, chief scientific officer and partner at C4 Laboratories, is currently researching some of the lesser-known molecules in cannabis, and he’s on to something. His research focuses on discovering new molecules, determining their therapeutic effects and expanding our understanding of the constituents of cannabis.

Dr. Zacariah Hildenbrand, chief scientific officer and partner at C4 Laboratories.
Dr. Zacariah Hildenbrand, chief scientific officer and partner at C4 Laboratories.

Dr. Hildenbrand received his Ph.D. from the University of Texas at El Paso where he researched the molecular architecture involved in hormone-dependent cancers. At the University of Texas Southwestern Medical Center in Dallas, his post-doctoral research contributed to the development of a novel therapy for the treatment of chronic myeloid leukemia, a blood-borne cancer that afflicts small children. He has published over 25 peer-reviewed scientific journal articles and hopes to do the same with his research in cannabis.

After a career of scientific consulting, Dr. Hildenbrand met Ryan Treacy, founder and chief executive officer of C4 Laboratories, in 2015 when Treacy launched the company. In June of 2015, the laboratory began operations, providing Dr. Hildenbrand the opportunity to embark on a new and exciting field of research- cannabis.

Ryan Tracy, founder and chief executive officer at C4 Labs.
Ryan Treacy, founder and chief executive officer at C4 Laboratories.

They currently collaborate with Dr. Kevin Schug of the Shimadzu Center for Advanced Analytical Chemistry (SCAAC) at the University of Texas, Arlington and together Drs. Schug and Hildenbrand are pursuing a DEA license to expand their current cannabis research. The SCAAC is a $10.0+ million analytical laboratory with instrumentation that only a handful of people in the world has access to.

C4 Laboratories, based in Mesa, Arizona, currently offers a range of services for cannabis analysis including terpene and cannabinoid analytics, microbial, pesticide, fungicide and insecticide testing. In addition to the standard gamut of tests, they also specialize in cultivation analytics like mold and mildew culture testing, viral detection with sentinel plants and comprehensive analysis of environmental conditions.

What makes their company unique is their multidisciplinary effort to characterize the therapeutic compounds found in cannabis, the C4 Cannabinomics Collaborative. We sit down with Dr. Zac Hildenbrand to talk cannabis science, his research and what they hope to accomplish with the C4 Cannabinomics Collaborative.

CannabisIndustryJournal: What is the C4 Cannabinomics Collaborative?

Dr. Zacariah Hildenbrand: The C4 Cannabinomics Collaborative is an open collaboration between growers and scientists to discover new molecules in cannabis and to have a better characterization of individual cannabis strains based on the active constituents found in each sample. We are facilitating the collaboration of some of the world’s best cannabis growers with world-class scientists to find new information about the plant.

What we want to accomplish in this work is identifying novel molecules. Because of the [federal government’s] restrictions in researching cannabis, there is very little peer-reviewed literature on many of the compounds found in cannabis. We want to secondarily find out what those molecules do in the human body and thus make recommendations for strains targeting specific conditions.c4 logo

We also want to understand the strains currently out there by determining the most established cannabinoids and terpenes via chemotyping. You hear a lot of people talking about the effects of an Indica or Sativa and making recommendations based on that. We want to find chemical signatures based on cannabinoids and terpenes and make recommendations based on that. There are a lot of problems at hand when discussing strain names scientifically. There are nomenclature issues- people calling the same strain different names, people giving multiple names to the same strain to make it appear that their strain portfolios are more diverse.

We can identify the chemical signatures in strains based on the major cannabinoids and terpenes. Based on the terpenes and chemical profile we can determine more accurate recommendations for patients as well as in recreational applications. All of this, again, discovering the new molecules, identifying the current strains, is so we can make more informed decisions regarding cannabis use. It is not a panacea but it is a very robust plant. There are a lot of terpenes with anti-inflammatory responses. Other molecules help with blood flow, sleep, regulating blood glucose, and we all know the cases of CBD helping children with convulsions and epilepsy. We want people to make sure they have the most up-to-date information.

CIJ: How is your collaboration with the SCAAC at UT Arlington contributing to this work?

Dr. Hildenbrand: One of the instruments we use there is a supercritical-fluid-extraction supercritical-fluid-chromatography mass-spectrometer (SFE-SFC-MS). With that instrument, we can do the extraction on the machine with an extreme level of sensitivity. It is ideal for drug discovery and identifying molecules in the parts-per-quadrillion range. This particular instrument allows us to detect molecules with an extreme level of sensitivity without volatizing them during the sample extraction process.

The Shimadzu Center for Advanced Analytical Chemistry
The Shimadzu Center for Advanced Analytical Chemistry

We want to acquire samples of unique cannabis from growers that will work with us to discover new cannabis constituents. We are in the process of getting a DEA license so that we can send products across state lines to the center at UT Arlington to perform the advanced characterization. They have instrumentation that only a handful of people in the world have access to, which gives us the best opportunity to explore the unknown. When we discover new molecules, find out what they do on the molecular level, we can then isolate these compounds and ultimately use this newfound knowledge for the development of effective nutraceuticals.

CIJ: What molecules are you researching right now?

Dr. Hildenbrand: Some of the low-hanging fruit in our research looks at identifying compounds similar to the better-studied compounds such as THC and CBD. THCV has a very similar structure to THC, but has a shorter acyl carbon chain (3 carbons vs. 5).

Tetrahydrocannabivarin (THCV)
Tetrahydrocannabivarin (THCV)

THCV doesn’t induce a psychoactive response (like THC), but it does improve fat utilization, so it has remarkable potential for medicine. We are looking at what conditions are required for it to occur naturally. Cannabis doesn’t produce THCV in a high amount. 0.7% by weight is the most we have seen in Arizona. In Oregon, where craft cannabis has been refined to a much higher degree, we have heard rumblings of some strains containing up to 3% THCV. We want to find out if this is a possible weight loss tool. Our research in CBDV is very much the same.Cannabinoid Biogenesis

CBL is the breakdown product of CBC when it is treated with ultraviolet light. We know absolutely nothing about what CBL does. If we find a strain that produces high amounts of CBC, we can then treat it with UV light and force the conversion to CBL, and then ultimately determine what it does. This is a good example of low-hanging fruit and the versatility of cannabis. Based on the biogenesis of the cannabinoids, we can alter the profile of cannabis products using a series of biochemical reactions.

Cannabicyclol (CBL)
Cannabicyclol (CBL)

For example, we have been helping clients in Arizona look for a quality sleep aid in cannabis. Certainly, Indica strains will help, but the molecule CBN helps specifically with sleep abnormalities. As CBN is formed as a byproduct when CBD or THC are oxidized, we see some producers using liquid nitrogen to oxidize CBD, leading to higher CBN levels. I would like to think we are in the age of understanding CBD, THC and the major terpenes,but there are a whole milieu of compounds that require our attention and THCV, CBDV and CBL are just a few that we want to devote our efforts to right away.

CIJ: What are your plans in the immediate future?

Dr. Hildenbrand: We are in the process of finalizing the documents to bring a C4 laboratory into Oregon where we can do quite a bit of research and where we’ll have access to some very unique cannabis. We will offer full compliance testing per ORELAP and OLCC regulations, but we also want to acquire samples (free of charge) from growers that want to collaborate with us to discover new molecules. We’ve been lucky enough to start working with growers like Adam Jacques and Chris West in Eugene, but we also want to be available to other growers who want to contribute to this research.

CIJ: What are your long-term goals with this project?

Dr. Hildenbrand: At a basic level, we hope to expand the current understanding of the cannabis plant. There is a lot of “bro science” and anecdotal claims out there. There is so much that we don’t know about cannabis that we cannot simply rely on anecdotal claims for each strain. We want to bring cannabis into the same light as any pharmaceutical-grade or biomedical research.

We need to be characterizing this plant with the same level of detail as other pertinent molecular therapies. In doing so there are a lot of potential discoveries to be made and we might be able to unlock the future of medicine. A drug like Marinol, for example, has been met with mixed reviews because its only one dimensional. Furthermore, we find that the terpene molecules are tremendously beneficial and this interplay between cannabinoids and terpenes is something that we want to explore further. All and all we wish to further illustrate the therapeutic capacities of cannabis within the contexts of specific ailments and medical conditions, while discovering the medicine of the future.