High levels of pesticides found in illicit cannabis inflorescence compared to licensed samples in Canadian study | Journal of Cannabis Research
Sampling
To reflect as realistically as possible the sources of cannabis inflorescence available to Canadians across the country, 36 licensed samples were purchased in 2021 from the Ontario Cannabis Store (Ontario, Canada) from licence holders located in all five Canadian regions (British Columbia, Prairies, Ontario, Quebec, and Atlantic) (Table 1). The 24 illicit cannabis samples were obtained from seizures by law enforcement officers across the country and submitted to Health Canada for laboratory testing in 2021.
Standards and reagents
Pesticide analytical standards were purchased from Chemservice (West Chester, PA) and Sigma-Aldrich Canada (Oakville, ON). Analytical grade acetone and toluene were purchased from EMD Millipore (Darmstadt, Germany). Analytical grade acetonitrile and Na2SO4 were purchased from Fisher Scientific (Fairlawn, NJ). Water was obtained from a Milli-Q® Plus Ultra Pure Water system (Millipore Corp., Burlington, MA). Sepra™C18-E was obtained from Phenomenex (Torrance, CA). Supelclean™ ENVI™-Carb SPE Tubes were obtained from Supelco (Bellefonte, PA). Sep-Pak® Classic NH2 Cartridges were obtained from Waters Corp. (Milford, MA).
Apparatus
For sample preparation, a laboratory blender 51BL30 (Stamford, Connecticut), a high-speed shaker (Spex Sample Prep Geno-Grinder; Fisher Scientific, Fairlawn, NJ), a centrifuge (Allegra X15R 208v; Beckman Coulter Inc., Brea, CA), a solvent evaporator (Xcelvap; Horizon Technologies, Salem, NH), and a rotary evaporator (Rotavpor R-114, BÜCHI Labortechnik AG, Flawil, Switzerland) were used. Sample analysis was carried out on a GC–MS/MS 7010B gas chromatograph quadrupole mass spectrometer/mass spectrometer (Agilent Technologies, Santa Clara, CA) and LC–MS/MS Exion HPLC 6500 Q-Trap triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA).
Standard solution preparation
High-concentration pesticide stock standard solutions were prepared from the purest analytical material commercially available, typically ≥ 95%. In general, stock standard solutions were prepared in the range of 1000–2500 μg/mL in acetone for GC–MS/MS compounds, and in either 100% acetonitrile or 100% methanol for LC–MS/MS compounds. From these, intermediate and spiking standard solutions were prepared respectively at 50 μg/mL and 1 μg/mL. Calibration standards were prepared with each sample set at concentrations of 0.8 × , 1 × , 2 × , 3 × , and 5 × the lowest calibrated level (LCL) in pesticide-free cannabis matrix extract to compensate for ion suppression/enhancement effects.
Sample preparation-dried cannabis flowers
Cannabis inflorescence samples (5–20 g) were homogenized in a laboratory blender. Acetonitrile (20 mL) was added to 2 g ground cannabis inflorescence sample and the mixture was extracted with a Geno-Grinder at 1750 rpm for 2 min. The tube was centrifuged at 4500 rpm for 5 min. Exactly 4 mL of the extract was added to a tube containing 1 g of dispersive C18 and shaken by Geno-Grinder at 1200 rpm for 1 min. Exactly 2 mL was transferred to an ENVI-Carb/Aminopropyl SPE containing 1 cm of Na2SO4, and eluted with 25 mL of 3:1 ACN:Toluene. The sample’s solvent was exchanged to acetone, blown down to less than 1 mL using a rotary evaporator, and 20 μL of 5 μg/mL 2,4,6-tribromobiphenyl was added as an internal standard. The sample was diluted to 1 mL with acetone. Half of the extract was transferred to a vial for GC–MS/MS analysis. The remaining portion’s solvent was exchanged to acetonitrile with solvent evaporator, brought to approximately 0.1 mL. Twenty microliters of isoprocarb 5 μg/mL was added as an internal standard, which was then diluted to 0.5 mL with acetonitrile and brought to 1 mL with H2O. The sample was filtered using a 1-cc plastic syringe and a 0.2-µm filter and transferred to a vial for LC–MS/MS analysis.
Instrument conditions
(a) LC–MS/MS—Sample analysis was carried out using a 6500 Q-Trap LC-MSMS (AB Sciex). Analyst version 1.6.3 (AB Sciex) and MultiQuant version 3.0.2 (AB Sciex) software were used for instrument control and data analysis, respectively. A Kinetex C18 column (2.1 × 50 mm, 2.6 μm) was used and maintained at 30 °C. The source was maintained at 550 °C. The following gas parameters were used: curtain gas, 35 psi; collision gas, 9psi; ion spray voltage, 5500 V; ion source gas 1, 50 psi; ion source gas 2, 55 psi. The injection volume was 1 μL. The mobile phases were water methanol (95 + 5) + 10 mM formic acid + 10 mM ammonium formate (A) and water–methanol (5 + 95) + 10 mM formic acid + 10 mM ammonium formate (B). The flow rate was 0.7 mL/min. The following elution gradient was used: 0–20 min, 0% B increasing to 100% B; 20–24.50 min, 100% B; 24.50–24.60 decreasing to 0% B then held from 24.60 to 25 min. Analysis was carried out by positive electrospray ionization using retention time-scheduled multiple reaction monitoring (MRM) to acquire two transitions (quantitative and qualitative) for each analyte. A partial list of these transition masses for both the LC − MS/MS and GC − MS/MS methods can be found in Table S1 and S2, respectively.
(b) GC–MS/MS—an Agilent 7010B GC–MS/MS carried out sample analysis. Mass Hunter software (Agilent) was used for instrument control and data analysis. The injection port was a multi mode injector (MMI) maintained at 250 °C. The liner was an inert double tapered splitless liner (Agilent # 5190–3983). The injection volume was 1 μL in splitless mode. Helium carrier gas was maintained at a constant flow of 1.0 mL/min. ZB-Multiresidue-1 capillary columns were used (2 columns; each of 15 m × 0.25 mm × 0.25 μm) (Phenomenex # 7EG-G016-11-CI) with backflush procedure at mid-column. The front column was fitted with a 1-m retention gap of the same stationary phase. The oven temperature was maintained at 60 °C for 1 min, ramped to 120 °C at 40 °C/min, then ramped to 310 °C at 5 °C/min with a 11.5-min hold (total run time: 52 min). The temperature of the MS source was maintained at 300 °C and the transfer line at 305 °C. Nitrogen was used as the collision gas at a flow of 1 mL/min. Analysis was carried out by electron impact ionization using dynamic MRM to acquire at least two transitions (quantitative and qualitative) for each analyte.
Validation criteria
Quantitative validation data must show that specific pesticide/matrix combinations can be accurately quantitated at the LCL deemed fit for purpose, the lowest value for the method being 0.01 µg/g. The LCL for each pesticide was determined by an injection of a series of matrix-matched standards. The LCL was deemed acceptable if the signal of the LCL peak height to the height of the surrounding noise was at a minimum of 5:1 ratio for two transitions for the GC–MS/MS and LC–MS/MS. This ratio is the relative intensity of the quantifying ion’s response compared to the qualifying ion’s response. Ion ratios must be within permitted tolerances to be acceptable (Table 2).
In addition, 5 replicate spikes at the LCL must meet method performance criteria of mean recoveries in the range of 70–120% with an RSD ≤ 20%. Exceptionally, a mean recovery below 70% may be acceptable if the recovery is consistent with an RSD ≤ 20% (European Commission, 2019).
The accuracy and precision of the pesticide recoveries were measured by spiking blank cannabis inflorescence matrix at the LCL (n = 5), 3 × LCL (n = 3) and 5 × LCL (n = 2). Linearity was established based on matrix-matched standards in the concentration range of 0.005–0.04 μg/mL for LC-MSMS, 0.010–0.080 μg/mL for GC-MSMS. The calibration curve generated from the standards must have a correlation coefficient (R2) greater or equal to 0.99.
Quality control
After the method was validated, samples were analysed with quality control measures in place for each sample set to ensure the integrity of the results. Each set of samples included a reagent blank, a matrix blank, and a representative matrix spike at the LCL for quality control. A blank sample was spiked with 200 µL of 0.1 µg/mL of GC–MS/MS and LC–MS/MS spiking solutions. The spike was allowed to stand for a minimum of 30 min. The blanks and spike were then processed the same way as the samples. To compensate for matrix effects on pesticides in plant material all standards were made from pesticide free cannabis inflorescences matrix extracts with the addition of pesticides standards at various concentrations. Results were calculated using a six-point calibration curve (at concentrations of 0.8 × , 1 × , 2 × , 3 × , 5 × , and 10 × the LCL).
