Last week, just before MJBizCon, PerkinElmer announced a collaboration with Emerald Scientific, allowing Emerald Scientific customers access to PerkinElmer’s portfolio of cannabis and hemp testing products and services. PerkinElmer is a leading instrument manufacturer and analytical method developer. Emerald Scientific is a distributor for scientific lab testing equipment and instrumentation.
Emerald Scientific now offers their customers PerkinElmer products, like their QSight® 420 Triple Quad system LC/MS, the Titan MPS™ Microwave Sample Preparation System, the Clarus® SQ 8 Gas Chromatograph/Mass Spectrometer (GC/MS) and the Flexar™ High-Performance Liquid Chromatography (HPLC) system. This partnership also allows Emerald Scientific customers to utilize the PerkinElmer analytical methods and standard operating procedures (SOPs) for cannabis and hemp testing. That includes SOPs for things like sample preparation, acquisition methods and consumable use. They’ll also be able to shop for lab products like PerkinElmer’s chromatography columns, vials and sample prep products.
According to Greg Sears, vice president and general manager, Food and Organic Mass Spectrometry at PerkinElmer, the cannabis testing market is exploding and this will help labs get their equipment and necessities all in the same place. “With the cannabis and hemp markets continuing to grow rapidly and regulations strengthening, labs increasingly need streamlined access to best-in-class, user-friendly testing solutions geared toward the unique requirements of the industry,” says Sears. ““This collaboration with Emerald Scientific brings together leading cannabis analysis offerings in one place to help labs start up and expand more efficiently. In addition, we can build on the work we have done with Emerald around testing standardization which is important for the science of the industry.”
Kirsten Blake, Vice President of Emerald Scientific, says they are really excited about the partnership. “As regulations become more challenging, laboratory competition intensifies, and the science of the industry receives increasing focus, it is essential to align with organizations dedicated to improving both the quality and throughput of analytics,” says Blake. “After working with PerkinElmer to inform, educate, and advance the cannabis science industry around best practices, we see them as the industry leader for providing analytical instrumentation, methods and SOP’s. By adding their complementary solutions to our existing portfolio, we can now deliver complete packaged analytical solutions to the cannabis and hemp industries.”
Back in August, Lake Superior State University (LSSU) announced the formation of a strategic partnership with Agilent Technologies to “facilitate education and research in cannabis chemistry and analysis.” The university formed the LSSU Cannabis Center of Excellence (CoE), which is sponsored by Agilent. The facility, powered by top-of-the-line Agilent instrumentation, is designed for research and education in cannabis science, according to a press release.
Chemistry student, Justin Blalock, calibrates an Agilent 1290 Ultra-High Pressure Liquid Chromatograph with a 6470 Tandem Mass Spectrometer in the new LSSU Cannabis Center of Excellence, Sponsored by Agilent.
The LSSU Cannabis CoE will help train undergraduate students in the field of cannabis science and analytical chemistry. “The focus of the new LSSU Cannabis CoE will be training undergraduate students as job-ready chemists, experienced in multi-million-dollar instrumentation and modern techniques,” reads the press release. “Students will be using Agilent’s preeminent scientific instruments in their coursework and in faculty-mentored undergraduate research.”
The facility has over $2 million dollars of Agilent instruments including their UHPLC-MS/MS, UHPLC-TOF, GC-MS/MS, LC-DAD, GC/MS, GC-FID/ECD, ICP-MS and MP-AES. Those instruments are housed in a 2600 square-foot facility in the Crawford Hall of Science. In February earlier this year, LSSU launched the very first program for undergraduate students focused completely on cannabis chemistry. With the new facility and all the technology that comes with it, they hope to develop a leading training center for chemists in the cannabis space.
Dr. Steve Johnson, Dean of the College of Science and the Environment at LSSU, says making this kind of instrumentation available to undergraduate studies is a game changer. “The LSSU Cannabis Center of Excellence, Sponsored by Agilent was created to provide a platform for our students to be at the forefront of the cannabis analytics industry,” says Dr. Johnson. “The instrumentation available is rarely paralleled at other undergraduate institutions. The focus of the cannabis program is to provide our graduates with the analytical skills necessary to move successfully into the cannabis industry.”
Storm Shriver is the Laboratory Director at Unitech Laboratories, a cannabis testing lab in Michigan, and sounds eager to work with students in the program. “I was very excited to learn about your degree offerings as there is a definite shortage of chemists who have experience with data analysis and operation of the analytical equipment required for the analysis of cannabis,” says Shriver. “I am running into this now as I begin hiring and scouting for qualified individuals. I am definitely interested in a summer internship program with my laboratory.”
LSSU hopes the new facility and program will help lead the way for more innovation in cannabis science and research. For more information, visit LSSU.edu.
Editor’s Note: The following is based on research and studies performed in their Santa Cruz Lab, with contributions from Mikhail Gadomski, Lab Manager, Ryan Maus, Technical Services Analyst, Dr. Laurie Post, Director of Food Safety & Compliance, Andy Sechler, Lab Director, Toby Astill, Senior Business Development Leader at Perkin Elmer and Charles Deibel, President of Deibel Cannabis Labs.
Pesticides represent the leading cause of batch failures in the cannabis industry. They are also the hardest tests to run in the laboratory, even one equipped with state-of-the-art equipment. The best instruments on the market are HPLC and GC dual mass spectrometer detectors, called “HPLC-qqq”, “GC-qqq,” or just triple quads.
As non-lab people, we envision a laboratory that can take a cannabis sample, inject it into a triple quad and have the machine quickly and effortlessly print out a report of pesticide values. Unfortunately, this is far from reality. The process is much more hands on and complex.In the current chemistry lab, trained analysts have to first program the triple quads to look for the pesticides of concern; in cannabis pesticide testing, this is done by programming the first of two mass spectrometers to identify a single (precursor) mass that is characteristic of the pesticide in question. For BCC requirements in California, this has to be done for all 66 pesticides, one at a time.
Next, these precursor ions are degraded into secondary chemicals called the “product” ions, also called transition ions. The second of the two mass spectrometers is used to analyze these transition ions. This process is graphed and the resulting spectrum is analyzed by trained chemists in the lab, pesticide by pesticide, for all the samples processed that day. If the lab analyzes 10 samples, that translates to 660 spectra to analyze (66 pesticides x 10 samples). When looking at the spectra for each pesticide, the analysts must compare the ratios of the precursor ions to the product ions.
Confirmation Testing
If these spectra indicate a given pesticide may be present, the chemists must then compare the ratios between the precursor and the products. If these ratios are not what is expected, then the analyst must perform confirmation testing to prove the precursor mass either is or is not the pesticide of concern. If the ratios are not what is expected, it means the molecule is similar to the pesticide in question, but may not be that pesticide. This confirmatory testing is key to producing accurate results and not failing batches when dealing with closely related chemicals. This process of analyzing spectra is done in all labs that are performing pesticide testing. In this fledgling industry, there are few published cannabis pesticide methods.
The need for this type of confirmation testing doesn’t happen all of the time, but when it does, it will take longer than our targeted three-day turn-around time. In the picture above, one precursor mass is ionized into several product masses; but only two are large enough to be used for comparison. In this hypothetical situation, two product masses are produced for every one precursor, the expected ion abundance ratio should be less than 30%. When performing any confirmatory testing, if the ion abundance ratio is >30%, it means the original precursor molecule was not the pesticide of concern. For example, if the ion abundance ratio was 50%, then the original molecule broke down into too many parts; it was not the pesticide we were looking for. This ion abundance ratio threshold was established by FANCO, the international organization that sets guidelines for all pesticide testing.
Testing Strategies
Methodology: In this fledgling industry, there are few published cannabis pesticide methods. The identification of the precursor mass and product ions are not always published, leaving labs to research which ions should be used. This adds to the potential for differences between lab results. Once selected, labs should validate their research, through a series of experiments to ensure the correct precursor and transition (product) ions are being used in the method.
Sample Preparation: Beyond the time-consuming work that is required to develop sound pesticide methods, the extraction step is absolutely critical for credible results. If the pesticides aren’t fully extracted from the cannabis product, then the results will be lower than expected. Sample preparations are often not standardized between labs, so unless a given extraction technique is validated for accuracy, there is the possibility for differences between labs.
Getting a Representative Sample
The current California recommended amount of sample is one gram of product per batch. Batch sizes can vary greatly and it is entirely likely that two different one gram samples can have two different results for pesticides. Has the entire plant been evenly coated with exactly the same amount of pesticide onto every square inch of its leaves? No, probably not. That is why it is imperative to take a “random” sample, by taking several smaller samples from different areas of the entire batch.
Sampling Plans: We can learn a lot from the manufacturing and sampling best practices developed by the food industry through the years. If a food manufacturer is concerned with the possibility of having a bacteria pathogen, like Salmonella, in their finished product, they test the samples coming off their production lines at a statistically relevant level. This practice (theory) is called the sampling plan and it can easily be adapted to the cannabis industry. The basic premise is that the more you test, the higher your likelihood of catching a contaminate. Envision a rectangular swimming pool, but instead of water, it’s filled with jello. In this gelatinous small pool, 100 pennies are suspended at varying levels. The pennies represent the contaminates.
Is the pool homogenized? Is jello evenly represented in the entire pool? Yes.
Is your concentrate evenly distributed in the extraction vessel? Yes. The question is, where are the pennies in that extraction vessel? The heavy metals, the microbial impurities and the pesticides should be evenly distributed in the extraction vessel but they may not be evenly represented in each sample that is collected. Unfortunately, this is the bane of the manufacturing industry and it’s the unfortunate reality in the food industry. If you take one random cup of jello, will you find the penny? Probably not. But it you take numerous 1 cup samples from random areas within the batch, you increase your chances of finding the contaminate. This is the best approach for sampling any cannabis product.
The best way to approve a batch of cannabis product is to take several random samples and composite them. But you may need to run several samples from this composite to truly understand what is in the batch. In the swimming pool example, if you take one teaspoon scoop, will you find one of the pennies? The best way to find one of the pennies is to take numerous random samples, composite them and increase the number of tests you perform at the lab. This should be done on any new vendor/cultivator you work with, in order to help establish the safety of the product.
By Dr. Zacariah Hildenbrand, Allegra Leghissa, Dr. Kevin A. Schug 2 Comments
Have you ever wondered why all beers have that strong, characteristic smell? Or why you could tell the smell of cannabis apart from any other plant? The answer is simple – terpenes.
These 55,000 different molecules are responsible for a majority of the odors and fragrances around us, from a pine forest, to the air diffuser in your house 1–3. They all share the same precursor, isoprene, and because of that, they are all related and have similar molecular structures. Unfortunately, it is this uncanny similarity that makes their analysis so challenging; we still lack a complete list of which terpenes expected to be found in each given plant species 1,2.
Many different methods have been developed in an effort to provide a time-optimized and straightforward analysis. Gas chromatography (GC) is usually center stage due to the volatility of the terpenes. Therefore, there is significant concern with the type of GC detector used 2.
The flame ionization detector (FID) is a good quantitative detector for GC, but qualitatively it does not provide any information, except for retention time; the differentiation between terpene species is achieved solely by use of retention indices (RI), which are based on elution times from a particular GC stationary phase. The best part of the FID is its low cost, reliability, and relatively easy interface, which make it an effective tool for quality control (QC) but less so with respect to research and discovery 2.
The primary choice for a research setting is the mass spectrometer (MS) detector. It is more expensive and complicated than FID, but importantly, it provides both good quantitative capabilities, and it provides mass spectra for each species that elutes from the chromatograph. However, for terpene analysis, it may still not be the best detector choice. Since terpene class molecules share many structural and functional similarities, even their fragmentation and sub-sequential identification by MS may lead to inconsistent results, which need to be confirmed by use of RI. Still, MS is a better qualitative analysis tool than the FID, especially for distinguishing non-isobaric terpenes 2.
Recently, new technology based on vacuum ultraviolet spectroscopy (VUV) has been developed as a new GC detector. The VUV detector enables analysis of virtually all molecules; virtually all chemical compounds absorb light in the range in the 125-240 nm wavelength range probed by the detector, making it an essentially universal detector 4–11. Previously, spectroscopic absorption detectors for GC have lacked sufficient energy to measure absorption of most GC-amenable species. The VUV detector fills a niche, which is complementary to MS detection in terms of the qualitative information it provides.
Figure 1: A, Section of the chromatographic separation of a terpenes standard mix; B, highlight of the co-eluting terpenes, camphor and (-)-isopulegol; C, differences in the absorbance spectra of camphor and (-)-isopulegol.
With the VUV detector, each compound exhibits its own unique absorbance spectrum. Even isomers and isobars, which are prevalent in terpene mixtures and can be difficult to distinguish different species by their electron ionization mass spectra, can be well differentiated based on their VUV spectra 6,9,10. Nevertheless, because analytes exhibit different spectra, it is not required to achieve a perfect chromatographic separation of the mixture components. Co-eluting peaks can be separated post-run through the use of library spectra and software inherent to the instrument 4,10. This ability is called “deconvolution”, and it is based on the fact that two co-eluting terpenes will give a peak with an absorbance spectrum equal to the sum of the two single absorbance spectra 4. Figure 1 shows the deconvolution process for two co-eluting terpenes, camphor and (-)-isopulegol. Due to their different absorbance spectra (Figure 1C), it is possible to fully separate the two peaks in post-run, obtaining sharp peaks for both analytes 6.
The deconvolution process has been shown to yield precise and accurate results. Thus, chromatographic resolution can be sacrificed in favor of spectroscopic resolution; this enables the development of methods with faster run times. With the ability to deconvolve unresolved peaks, a long temperature ramp to chromatographically separate all isomeric terpenes is not required 6. Additionally, the presence of coeluting components, which might normally go undetected with some GC detectors, can be easily judged based on comparison of the measured spectra with pure reference spectra contained in the VUV spectral library.
The other issue in terpenes analysis is the extraction process. Terpenes can be extracted with the use of solvents (e.g., methanol, ethanol, hexane, and cyclohexane, among others), but the process is usually time-consuming, costly and not so environmentally-friendly 2. The plant needs to be manually crushed and then aliquots of solvent are used to extract components from the plant, ideally at least 3 times and combined to achieve acceptable results. The problem is that some terpenes may respond better to a certain solvent, making their extraction easier and more optimized than for others 2. The choice of solvent can cause discrimination against the extraction some terpenes, which limits the comprehensiveness of analysis.
Headspace is another technique that can be used for the sample preparation of terpenes. Headspace sampling is based on heating the solid or liquid sample inside a sealed vial, and then analyzing the air above it after sufficient equilibration. In this way, only volatile analytes are extracted from the solid/liquid sample into the gas phase; this allows relatively interference-free sampling 12–14.
How do we know whether our extraction analysis methods are correct and comprehensive for a certain plant sample? Unfortunately, there is not a complete list of available molecules for each plant species, and even if two specimens may smell really similar to our nose, their terpenes profiles may be notably different. When working with a new plant material, it is difficult to predict the extraction efficiency for the vast array of terpenes that may be present. We can only perform it with different extraction and detection methods, and compare the results.
The route for a comprehensive and fast analysis of terpenes is therefore still long; however, their intoxicating aromas and inherent medicinal value has provided a growing impetus for researchers around the world. Considering the evolving importance of Cannabis and the growing body of evidence on the synergistic effects between terpenes and cannabinoids, it is likely that newly improved extraction and analysis methods will be developed, paving the way for a more complete list of terpene species that can be found in different cultivars. The use of new analytical technologies, such as the VUV detector for GC, should aid considerably in this endeavor.
References:
[1] Breitmaier E., Terpenes: Flavors, Fragrances, Pharmaca, Pheromones. John Wiley & Sons 2006.
[2] Leghissa A., Hildenbrand Z. L., Schug K. A., A Review of Methods for the Chemical Characterization of Cannabis Natural Products. J. Sep. Sci.2018, 41, 398–415 .
[3] Benvenuto E., Misra B. B., Stehle F., Andre C. M., Hausman J.-F., Guerriero G., Cannabis sativa: The Plant of the Thousand and One Molecules. Front. Plant Sci2016, 719, DOI: 10.3389/fpls.2016.00019.
[4] Schug K. A., Sawicki I., Carlton D. D., Fan H.,Mcnair H. M.,Nimmo J. P., Kroll P.,Smuts J.,Walsh P., Harrison D., Vacuum Ultraviolet Detector for Gas Chromatography. Anal. Chem.2014, 86, 8329–8335 .
[5] Fan H.,Smuts J., Walsh P.,Harrison D., Schug K. A., Gas chromatography-vacuum ultraviolet spectroscopy for multiclass pesticide identification. J. Chromatogr. A2015, DOI: 10.1016/j.chroma.2015.02.035.
[6] Qiu C.,Smuts J., Schug K. A., Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J. Sep. Sci.2017, 40, 869–877 .
[7] Leghissa A., Smuts J., Qiu C., Hildenbrand Z. L., Schug K. A., Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Sep. Sci. Plus2018, 1.
[8] Bai L.,Smuts J., Walsh P., Fan H., Hildenbrand Z., Wong D., Wetz D., Schug K. A., Permanent gas analysis using gas chromatography with vacuum ultraviolet detection. J. Chromatogr. A2015,1388, 244–250 .
[9] Skultety L., Frycak P., Qiu C.,Smuts J., Shear-Laude L., Lemr K., Mao J. X., Kroll P., Schug K. A., Szewczak A., Vaught C., Lurie I., Havlicek V., Resolution of isomeric new designer stimulants using gas chromatography – Vacuum ultraviolet spectroscopy and theoretical computations. Anal. Chim. Acta2017, 971, 55–67 .
[10] Bai L., Smuts J., Walsh P., Qiu C., McNair H. M., Schug K. ., Pseudo-absolute quantitative analysis using gas chromatography–vacuum ultraviolet spectroscopy–a tutorial. Anal. Chim. Acta2017, 953, 10–22 .
[11] Schenk J., Nagy G., Pohl N. L. B., Leghissa A., Smuts J., Schug K. A., Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy. J. Chromatogr. A2017, 1513, 210–221 .
[12] Van Opstaele F., De Causmaecker B., Aerts G., De Cooman L., Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry. J. Agric. Food Chem.2012, 60, 12270−12281 .
[13] Hamm S., Bleton J., Connan J., Tchapla A., A chemical investigation by headspace SPME and GC-MS of volatile and semi-volatile terpenes in various olibanum samples. Phytochemistry2005,66, 1499–1514 .
[14] Aberl A., Coelhan M., Determination of volatile compounds in different hop Varieties by headspace-trap GC/MS-in comparison with conventional hop essential oil analysis. J. Agric. Food Chem.2012, 60, 2785−2792 .
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Emerald Scientific now offers their customers PerkinElmer products, like their QSight® 420 Triple Quad system LC/MS, the Titan MPS™ Microwave Sample Preparation System, the Clarus® SQ 8 Gas Chromatograph/Mass Spectrometer (GC/MS) and the Flexar™ High-Performance Liquid Chromatography (HPLC) system. This partnership also allows Emerald Scientific customers to utilize the PerkinElmer analytical methods and standard operating procedures (SOPs) for cannabis and hemp testing. That includes SOPs for things like sample preparation, acquisition methods and consumable use. They’ll also be able to shop for lab products like PerkinElmer’s chromatography columns, vials and sample prep products.
According to Greg Sears, vice president and general manager, Food and Organic Mass Spectrometry at PerkinElmer, the cannabis testing market is exploding and this will help labs get their equipment and necessities all in the same place. “With the cannabis and hemp markets continuing to grow rapidly and regulations strengthening, labs increasingly need streamlined access to best-in-class, user-friendly testing solutions geared toward the unique requirements of the industry,” says Sears. ““This collaboration with Emerald Scientific brings together leading cannabis analysis offerings in one place to help labs start up and expand more efficiently. In addition, we can build on the work we have done with Emerald around testing standardization which is important for the science of the industry.”
