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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.

Oregon Issues Health Alert for Contaminated Cannabis

By Aaron G. Biros
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According to Jonathan Modie, spokesman for the Oregon Health Authority (OHA), on Friday, October 21st, the OHA issued a ‘health alert’ regarding cannabis products sold from a McMinnville dispensary that were possibly tainted with extremely high levels of Spinosad, an insecticide commonly used to combat mites and other pests. “My understanding is that two medical patients purchased the cannabis products whom we had contact info for, but most of the purchasers were recreational customers,” says Modie. “Because it is not required to get contact info for recreational customers, we issued the health alert to get the word out as quickly as possible because we didn’t know who bought the product.” The OHA is urging consumers who purchased cannabis from New Leaf CannaCenter in McMinnville to check the labels and see if they purchased potentially dangerous cannabis, and to either return the cannabis to the dispensary or dispose of it appropriately.

oha_logo_lrgThe action level, the measured amount of pesticides in a product that the OHA deems potentially dangerous, for Spinosad is 0.2 parts-per-million (PPM). The two batches in question are the strains Dr. Jack (batch number G6J0051-02) and Marion Berry (batch number G6J0051-01), which were tested to contain approximately 42 PPM and 22 PPM respectively, much higher than the OHA’s action level.

While this is the first health alert issued in Oregon in connection with potentially contaminated cannabis, Modie says he expects there will be more health alerts in the future. “Unfortunately the product was inappropriately transferred from the grower to the dispensary and from the dispensary to customers, so we are working to get the word out to dispensaries, growers and processors about the testing rules to prevent this from happening in the future,” says Modie. “We want to make it clear that any grower, processor or dispensary that does not follow the testing requirements or fail to label, store or retain batches that fail a test will be subject to enforcement actions such as fines, penalties, suspension or revocation of their license.” The OHA has a list of pesticide analytes and their action levels on their website.

“We are advising recreational and medical users alike to read the product labels closely; the labels must have the license or registrant number, the packaging or distributor license number, the name of the strain and the universal symbol,” says Modie. “We are also suggesting consumers request a copy of pesticide test results from the dispensary.” It is unclear at this time if all of the cannabis products in question have been properly disposed of, but OHA was informed that New Leaf has pulled all products in question off of the shelf.