Pesticide analysis for cannabis flower: method and data overview

Sara in her Restek Chromatography Mastermind shirt.

Pesticides in cannabis has been a hot topic lately and we have been getting many requests for help with this analysis. We did work on pesticide residue testing in cannabis about 6 years ago. At that time, we were limited to testing our methods with a small amount of seized material. Recently, we have been able to work with great collaborators, Shimadzu Scientific Instruments and Trace Analytics, to do more comprehensive method development and partial validation. Trace Analytics is a testing laboratory servicing the medical and recreation cannabis inustry in Spokane, Washington since 2015. With the help of our collaborators and availability of bulk samples, we were able to revisit method development and perform a partial validation. This would not have been possible without the hard work of all of the team members; Jeff Dahl, Caitlin Johnson, Derek Laine, Sara Minier, Amanda Rigdon, Gordon Fagras, and  Jack Cochran.

Amanda and Derek doing some creative plumbing

Sara, Caitlin and Julie too busy to look at the camera.

We developed a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation method (stay tuned for a more discussion in a future blog) paired with LC-MS/MS analysis using a Shimadzu LCMS-8050 with Prominence HPLC. QuEChERS is designed to be generic and work for a wide variety of pesticides with diverse chemical properties. This is one of the reasons why QuEChERS is so popular for food safety testing. This approach takes advantage of the selectivity and sensitivity of LC-MS/MS allowing us to use a “good enough” sample preparation. QuEChERS is much less intensive than what would be needed if a non-MS/MS based method was used…another reason why it is popular.

We used a 1 µL injection to help with early eluting pesticides peak shapes since the extraction solvent is acidified acetonitrile and the initial mobile phase is mainly water. This also helps maintain column and instrument cleanliness allowing many injections to be made before maintenance is needed. At least two MS/MS transitions were monitored for each analyte.

Take a look at the method details of our sample prep protocol and analysis method.

We tested three different cannabis flower matrices. They included orange kush flower, permafrost flower and then a composite. The composite sample was simply a combination of flower and sugar leaves from several strains. We did this so we had enough “pesticide-free” sample to perform all of the spiking experiments.  Each matrix was spiked at three levels (50, 200, 1000 µg/kg “dry” weight) with the Restek Oregon pesticide standard (more information in "sample prep protocol" page 2). These levels were chosen based on current and proposed regulatory limits from various states. Each spike level was performed in triplicate. The spiking scheme is shown in the table below. Triplicate data allows us to determine average recovery and relative standard deviation (RSD) for each level in each matrix as well as across the three matrices. Matrix-matched calibration was used for quantitation and both method and instrument internal standards were used.

 

1.5 grams flower with water and acetonitrile

 

The data shown here is for “dry” flower material and so we used a reduced sample amount (1.5 g) and a hydration step. Adding water is critical for the extraction chemistry to work properly. The AOAC QuEChERS extraction salts were used for the salting out step of the extraction. After the extraction step, we chose to use the “universal” formulation of the dispersive solid phase extraction (dSPE) to remove unwanted coextracted compounds. This formula contains 50 mg/mL extract each of primary secondary amine (PSA) and octadecyl (C18) sorbents and a moderate level of graphitized carbon black (GCB) (7.5 mg GCB per mL of extract). GCB removes chlorophyll which can cause instruments to become dirty if injected.

For more information on QuEChERS, see this blog QuEChERS – Where to start? and others on our website.

Take a look at recovery values and RSDs table.

Highlights of the data:

• For the large majority of pesticides, in all three matrices AND at all three spike levels, recovery was between 70-120% which is the desirable range for the food safety industry. It is expected that difficult pesticides or detection at very low levels may produce recovery below this range. In most of these cases, RSD values were less than 20% for a single matrix (n=9) and across all three matrices (n=27). This data indicates that this method is appropriate for multi-residue pesticide analysis in cannabis flower.
• There are some of the Oregon pesticides that are more amenable to GC analysis and we would suggest using GC-MS/MS testing of the extracts to cover bifenthrin, cyfluthrin, dichlorvos, MGK-264, and permethrin. We were able to determine recovery at the highest spike level (1000 ppb) with LC-MS/MS for bifenthrin, dichlorvos, MGK-264 and they fell within the 70-120% recovery and less than 20% RSD so this indicates that the sample preparation method is suitable.
• Abamectin, widely known by the trade name Avid, is a popular insecticide used to treat spider mites which are known to attack cannabis plants. Abamectin is heat sensitive and so the hot LC-MS/MS interface used for other pesticides caused the abamectin signal to be low. Abamectin can be tested with different LC-MS/MS interface parameters to obtain maximum signal and detect it a low levels. However, even using the multi-residue method, we were able to confirm recovery of 85% (7% RSD, n= 9) for the 1000 ppb level.
• Spiromesifen is an insensitive compound and detectability could be improved by increasing the injection volume for LC-MS/MS or by using GC-MS/MS analysis.
• Spinosad and spiroxamine show slightly lower recovery values ranging from 60% and higher but these recovery values are generally consistent with less than 20% RSD across all spike levels and matrices (only one exception). These compounds are slightly basic and recovery may be lower than other pesticides because acidic extraction conditions are used during sample preparation.
• Overall, this method is a great approach for multi-residue pesticide testing in cannabis as demonstrated by the acceptable recovery of nearly all of the almost 60 pesticides we tested.

Stay tuned for more information and details about pesticide analysis in cannabis.