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dSPE cleanups

The Grass Isn’t Always Greener: Removal of Purple Pigmentation from Cannabis

By Danielle Mackowsky
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dSPE cleanups
Cannabis strains used (clockwise from top left): Agent Orange, Tahoe OG, Blue Skunk, Grand Daddy and Grape Drink

Cannabis-testing laboratories have the challenge of removing a variety of unwanted matrix components from plant material prior to running extracts on their LC-MS/MS or GC-MS. The complexity of the cannabis plant presents additional analytical challenges that do not need to be accounted for in other agricultural products. Up to a third of the overall mass of cannabis seed, half of usable flower and nearly all extracts can be contributed to essential oils such as terpenes, flavonoids and actual cannabinoid content1. The biodiversity of this plant is exhibited in the over 2,000 unique strains that have been identified, each with their own pigmentation, cannabinoid profile and overall suggested medicinal use2. While novel methods have been developed for the removal of chlorophyll, few, if any, sample preparation methods have been devoted to removal of other colored pigments from cannabis.

Cannabis samples following QuEChERS extraction

Sample Preparation

Cannabis samples from four strains of plant (Purple Drink, Tahoe OG, Grand Daddy and Agent Orange) were hydrated using deionized water. Following the addition of 10 mL acetonitrile, samples were homogenized using a SPEX Geno/Grinder and stainless steel grinding balls. QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) non-buffered extraction salts were then added and samples were shaken. Following centrifugation, an aliquot of the supernatant was transferred to various blends of dispersive SPE (dSPE) salts packed into centrifugation tubes. All dSPE tubes were vortexed prior to being centrifuged. Resulting supernatant was transferred to clear auto sampler vials for visual analysis. Recoveries of 48 pesticides and four mycotoxins were determined for the two dSPE blends that provided the most pigmentation removal.

Seven dSPE blends were evaluated for their ability to remove both chlorophyll and purple pigmentation from cannabis plant material:

  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg C18, 50 mg Chlorofiltr®
  • 150 mg MgSO4, 50 mg PSA
  • 150 mg MgSO4, 25 mg C18
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18
  • 150 mg MgSO4, 25 mg PSA, 7.5 mg GCB
  • 150 mg MgSO4, 50 mg PSA, 50 mg C18, 50 mg GCB

Based on the coloration of the resulting extracts, blends A, F and G were determined to be the most effective in removing both chlorophyll (all cannabis strains) and purple pigments (Purple Drink and Grand Daddy). Previous research regarding the ability of large quantities of GCB to retain planar pesticides allowed for the exclusion of blend G from further analyte quantitation3. The recoveries of the 48 selected pesticides and four mycotoxins for blends A and F were determined.

dSPE cleanups
Grand Daddy following various dSPE cleanups


A blend of MgSO4, C18, PSA and Chlorofiltr® allowed for the most sample clean up, without loss of pesticides and mycotoxins, for all cannabis samples tested. Average recovery of the 47 pesticides and five mycotoxins using the selected dSPE blend was 75.6% were as the average recovery when including GCB instead of Chlorofiltr® was 67.6%. Regardless of the sample’s original pigmentation, this blend successfully removed both chlorophyll and purple hues from all strains tested. The other six dSPE blends evaluated were unable to provide the sample clean up needed or had previously demonstrated to be detrimental to the recovery of pesticides routinely analyzed in cannabis.


(1)           Recommended methods for the identification and analysis of cannabis and cannabis products, United Nations Office of Drugs and Crime (2009)

(2)            W. Ross, Newsweek, (2016)

(3)            Koesukwiwat, Urairat, et al. “High Throughput Analysis of 150 Pesticides in Fruits and Vegetables Using QuEChERS and Low-Pressure Gas Chromatography Time-of-Flight Mass Spectrometry.” Journal of Chromatography A, vol. 1217, no. 43, 2010, pp. 6692–6703., doi:10.1016/j.chroma.2010.05.012.


10 Treatment Methods to Reduce Mold in Cannabis

By Ketch DeGabrielle

As the operations manager at Los Sueños Farms, the largest outdoor cannabis farm in the country, I was tasked with the challenge of finding a yeast and mold remediation treatment method that would ensure safe and healthy cannabis for all of our customers while complying with stringent regulations.

While outdoor cannabis is not inherently moldy, outdoor farms are vulnerable to changing weather conditions. Wind transports spores, which can cause mold. Each spore is a colony forming unit if plated at a lab, even if not germinated in the final product. In other words, perfectly good cannabis can easily fail microbial testing with the presence of benign spores.

Fun Fact: one square centimeter of mold can produce over 2,065,000,000 spores.

If all of those landed on cannabis it would be enough to cause over 450 pounds of cannabis to fail testing, even if those spores remained ungerminated.

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

It should also be known that almost every food item purchased in a store goes through some type of remediation method to be considered safe for sale. Cannabis is finally becoming a legitimized industry and we will see regulations that make cannabis production look more like food production each year.

Regulations in Colorado (as well as Nevada and Canada) require cannabis to have a total yeast and mold count (TYMC) of ≤ 10,000 colony forming units per gram. We needed a TYMC treatment method that was safe, reliable, efficient and suitable for a large-scale operation. Our main problem was the presence of fungal spores, not living, growing mold.

Below is a short list of the pros and cons of each treatment method I compiled after two years of research:

Autoclave: This is the same technology used to sterilize tattoo needles and medical equipment. Autoclave uses heat and pressure to kill living things. While extremely effective, readily available and fiscally reasonable, this method is time-consuming and cannot treat large batches. It also utilizes moisture, which increases mold risk. The final product may experience decarboxylation and a change in color, taste and smell.

Dry Heat: Placing cannabis in dry heat is a very inexpensive method that is effective at reducing mold and yeast. However, it totally ruins product unless you plan to extract it.

An autoclave
Image: Tom Beatty, Flickr

Gamma Ray Radiation: By applying gamma ray radiation, microbial growth is reduced in plants without affecting potency. This is a very effective, fast and scalable method that doesn’t cause terpene loss or decarboxylation. However, it uses ionizing radiation that can create new chemical compounds not present before, some of which can be cancer-causing. The Department of Homeland Security will never allow U.S. cannabis farmers to use this method, as it relies on a radioactive isotope to create the gamma rays.

Gas Treatment: (Ozone, Propylene Oxide, Ethylene Oxide, Sulfur Dioxide) Treatment with gas is inexpensive, readily available and treats the entire product. Gas treatment is time consuming and must be handled carefully, as all of these gases are toxic to humans. Ozone is challenging to scale while PPO, EO and SO2 are very scalable. Gases require special facilities to apply and it’s important to note that gases such as PPO and EO are carcinogenic. These methods introduce chemicals to cannabis and can affect the end product by reducing terpenes, aroma and flavor.

Hydrogen Peroxide: Spraying cannabis plants with a hydrogen peroxide mixture can reduce yeast and mold. However, moisture is increased, which can cause otherwise benign spores to germinate. This method only treats the surface level of the plant and is not an effective remediation treatment. It also causes extreme oxidation, burning the cannabis and removing terpenes.

Microwave: This method is readily available for small-scale use and is non-chemical based and non-ionizing. However, it causes uneven heating, burning product, which is damaging to terpenes and greatly reduces quality. This method can also result in a loss of moisture. Microwave treatment is difficult to scale and is not optimal for large cultivators.

Radio Frequency: This method is organic, non-toxic, non-ionizing and non-chemical based. It is also scalable and effective; treatment time is very fast and it treats the entire product at once. There is no decarboxylation or potency loss with radio frequency treatment. Minimal moisture loss and terpene loss may result. This method has been proven by a decade of use in the food industry and will probably become the standard in large-scale treatment facilities.

Steam Treatment: Water vapor treatment is effective in other industries, scalable, organic and readily available. This method wets cannabis, introducing further mold risk, and only treats the product surface. It also uses heat, which can cause decarboxylation, and takes a long time to implement. This is not an effective method to reduce TYMC in cannabis, even though it works very well for other agricultural products

extraction equipment
Extraction can be an effective form of remediating contaminated cannabis

Extraction: Using supercritical gas such as butane, heptane, carbon dioxide or hexane in the cannabis extraction process is the only method of remediation approved by the Colorado Marijuana Enforcement Division and is guaranteed to kill almost everything. It’s also readily available and easy to access. However, this time-consuming method will change your final product into a concentrate instead of flower and usually constitutes a high profit loss.

UV Light: This is an inexpensive and readily available method that is limited in efficacy. UV light is only effective on certain organisms and does not work well for killing mold spores. It also only kills what the light is touching, unless ozone is captured from photolysis of oxygen near the UV lamp. It is time consuming and very difficult to scale.

After exhaustively testing and researching all treatment methods, we settled on radio frequency treatment as the best option. APEX, a radio frequency treatment machine created by Ziel, allowed us to treat 100 pounds of cannabis in an hour – a critical factor when harvesting 36,000 plants during the October harvest.

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.


How Potent is Your Product: Getting Educated on Edibles Analysis

By Danielle Mackowsky
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As a result of the rapidly developing cannabis industry, many forensic toxicology labs are looking for fast, reliable and cost-effective methods to determine cannabis potency and pesticide residue in edibles. Although the pros and cons of legalization are still heavily debated throughout the country, all scientists agree that uniform testing policies and procedures need to be established as soon as possible.

Within environmental and food testing laboratories, the use of QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) has been practiced widely for the past 15 years. In 2003, Dr.’s Michelangelo Anastassiades and Steven Lehotay published the first QuEChERS application, which detailed the determination of pesticide residues in produce. Since then, QuEChERS has become the gold standard for the testing and analysis of a wide variety of edible matrices. United Chemical Technologies (UCT) was the first company to commercialize the product and it became apparent that the application of this technology to cannabis edibles was a natural solution to pesticide residue testing. All of the data from the QuEChERS cannabis edibles pesticide and potency analyses can be found here.

Sample preparation

Hard candy before freezer mill grinding

Preparation of a sample for QuEChERS analysis varies depending on the type of edible product being tested. Baked goods, chocolate bars and hard candies should be ground into a fine powder prior to analysis. Although this can be achieved using a product such as a SPEX 6770 freezer mill, a blender can suffice when analyzing typical plant-based samples. Liquid samples, such as sodas or teas, should be degassed prior to analysis, whereas any gummy-based candies should be cut into fine pieces. With the exception of the liquid samples, all other matrices should then be hydrated for one hour within a QuEChERS extraction tube.

Hard candy after freezer mill grinding

Following sample preparation, acetonitrile is added to all samples along with a proprietary blend of QuEChERS extraction salts. These salts remove water from the organic phase, help to facilitate solvent partitioning and protect base-sensitive analytes from degradation. After shaking and centrifuging the sample, three distinct layers are formed. The top layer, which is the organic phase, can then be aliquoted off for further sample clean-up or dilution.

A mint milk chocolate sample after QuEChERS extraction

For pesticide analysis, an aliquot of the organic layer was subjected to dispersive solid phase extraction (dSPE). This process utilizes an additional blend of proprietary sorbents that remove chlorophyll, sugars, organic acids and fatty compounds from the sample. The resulting extract is free of pigmentation and is ready for analysis on the LC-MS/MS. All samples that were analyzed for cannabinoids did not undergo dSPE; rather, a serial dilution was carried out due to the high concentration of cannabinoids in the original organic layer. The original QuEChERS extract required a dilution of 100-200x in order to have a sample that was ultimately suitable for analysis on LC-MS/MS. A UCT Selectra Aqueous C18 HPLC Column and Guard Column were used in a Thermo Scientific Dionex UltiMate 3000 LC System. An aqueous C18 column was selected due to the extreme polarity of the pesticides being analyzed.

Comparison of QuEChERS extracts before and after dSPE cleanup (gummy sample)


This application utilizes the advantages of  UCT’s proprietary QuEChERS combination to extract 35 pesticides and 3 cannabinoids, including tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in edibles, followed by either serial dilutions for cannabis potency analysis, or a dSPE cleanup for pesticide residue analysis. This hybrid method allows QuEChERs, which are extensively used in the food testing industry, to be utilized in a forensic setting.