A Novel Comprehensive Strategy for Residual Pesticide Analysis in Cannabis Flower
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Application Note Cannabis Testing, Food A novel comprehensive strategy for residual pesticide analysis in cannabis flower Authors Abstract Lilly Asanuma1, Dan Miller1, Eight U.S. states have approved the recreational sale of cannabis, with an additional Rick Jordan1, Melissa Churley2, 35 states allowing some degree of medical use, or use of cannabidiol‑containing and Anthony Macherone2,3 products for limited medicinal purposes. Every state that has legalized use in 1 Pacific Agricultural some form has testing requirements for pesticide residues in cannabis flower and Laboratory, cannabis products before retail sale. Each state has its own testing requirements Sherwood, OR 97140 regarding the specific pesticides to be evaluated, and the action levels that the 2 Agilent Technologies, Inc. tested pesticides must not exceed. Pacific Agricultural Laboratory has validated Santa Clara, CA 95051 workflows using LC/MS/MS and GC/MS/MS to cover these different requirements across a range of products. This Application Note describes a comprehensive 3 Johns Hopkins University pesticide screening and quantitation approach for up to 214 pesticide residues School of Medicine in cannabis flower. The methodology uses a single extraction and combined analyses using the Agilent 7010 Tandem Quadrupole and Agilent 6470 Tandem Quadrupole Mass Spectrometer systems. For GC/MS/MS, limits of quantitation (LOQs) were determined to be 0.1 mg/kg for 94 % of the comprehensive target list. For LC/MS/MS, LOQs were determined to be 0.1 mg/kg for 89 % of the comprehensive target list. Analyte recoveries were between 70–120 % for 212 of the 214 compounds. Introduction The protocol was based on the sample Source. This efficiency permits high preparation procedure used to determine sample dilution factors that minimize Quantifying pesticide residues in pesticides in dried hops1 with an matrix effects while still achieving the cannabis flower is a complex problem. additional dilution step before analysis. required limits of quantitation (LOQs), The challenge is partially due to the The dilute eluate is then passed to both determined to be 0.1 mg/kg for 94 % of concentration level disparities between GC/MS/MS and LC/MS/MS workflows, the comprehensive target list. naturally occurring cannabinoids, as described below, for comprehensive To achieve the necessary separations, incurred pesticide residues, and other pesticide residue analysis. More than the LC/MS/MS system used an endogenous compounds such as 200 known pesticides were tested, going Agilent InfinityLab Poroshell 120 terpenes, which typically are found at far beyond the often‑referenced Oregon Phenyl-Hexyl column (2.1 × 100 mm, concentrations of 1 to 2 % by weight of list of approved pesticides for cannabis. 2.7 μm, p/n 695775-912). An online the flower. The typical extraction process To help minimize matrix effects and multisampler pretreatment procedure has the potential for low pesticide provide acceptable recoveries for each was used to improve the peak shape of recoveries and deleterious effects on target list, dispersive SPE cleanup (dSPE) early eluting compounds. This procedure the analytical instrumentation caused techniques were developed for each entails sandwiching the sample between by co-extracted material. Our approach platform. HPLC grade water using the sandwiched to sample preparation exploits the The GC/MS/MS system was equipped injection features of the autosampler. benefits of highly sensitive instruments, with two columns of differing polarities, The sensitivity of the Agilent 6470 allowing for significant sample dilution to and used midcolumn backflush with MS/MS system allows for high dilution decrease these matrix effects. the Agilent Purged Ultimate Union. The factors that minimize matrix effects, An acetonitrile extraction of the cannabis Agilent 7010 GC/MS/MS includes a High and achieve the required LOQs. For extract was used, followed by passing Efficiency Source (HES), which results in the comprehensive target list, 89 % of the extract through a SPE cartridge. the creation of up to 20‑times more ions the compounds achieved an LOQ of compared to the Standard Agilent Inert 0.1 mg/kg. 2 Experimental Weigh 1.0 g of sample into a 50-mL centrifuge tube. Sample preparation Add 15 mL of acetonitrile to each tube. Figure 1 shows the sample preparation workflow for GC/MS/MS and LC/MS/MS. Add ceramic homogenizer, cap the tube, and First, 1.0 g of homogenized cannabis mechanically shake it for 2 minutes (1,500 strokes/min). flower was accurately weighed into 50-mL centrifuge tubes. Then, 15 mL of Decant the solvent into a unconditioned SPE cartridge high‑purity, pesticide‑grade acetonitrile (recommend SampliQ C18 Endcapped p/n 5982-1365), and collect the eluent. was added to each tube. The tubes were sealed and mechanically shaken Rinse the centrifuge tube twice with 5 mL of acetonitrile, for 2 minutes at 1,500 strokes/minute. passing through the SPE, transferring all solids with second rinse. The extract was passed through a unconditioned polymeric solid phase Bring the collected eluent to a 25 mL final volume with acetonitrile (25-fold dilution). extraction (SPE) cartridge and eluted by gravity. The centrifuge tube was rinsed twice with 5 mL of acetonitrile, GC/MS/MS LC/MS and passed through the SPE cartridge. During the final rinse, the sample solids Add 100 µL of extract + 900 µL of Mix 50 µL extract + LC/MS/MS cleanup were transferred to the SPE cartridge. All hexane/acetone (1:1) to a 2-mL dSPE 950 µL acetonitrile (if necessary)* (500-fold dilution). fractions were collected and brought to a cartridge (p/n 5982-0028) (250-fold dilution). final volume of 25 mL with acetonitrile. Add 1,000 µL of the Samples are ready for extract to a 2-mL Vortex the tube for 30 seconds, then LC/MS/MS analysis. dSPE cartridge centrifuge for 2 minutes at 2,000 rpm. (p/n 5982-5122). Add 300 µL of supernatant + 300 µL of Vortex the cartridge for hexane/acetone (1:1 v/v) 30 seconds, then (500-fold dilution). centrifuge for 2 minutes at 2,000 rpm. Samples are ready for GC/MS/MS analysis. Mix 50 µL of the supernatant with 950 µL of acetonitrile (500-fold dilution). Samples are ready for LC/MS/MS analysis. * An optional cleanup step can be used for LC/MS/MS compounds if necessary (that is, the observation of matrix interference, poor chromatography, and so forth, when using the simpler dilution approach) using the Agilent dispersive-SPE kit (p/n 5982-5122). In this procedure, 1 mL of collected sample extract is added to a 2-mL d-SPE cartridge, vortexed/centrifuged, and diluted 20-fold (50 µL treated extract + 950 µL acetonitrile) prior to analysis. Please note, PSA is a powerful cleanup sorbent for cannabis analysis, and Daminozide, Spinosad, Spirotetramat, and Spiroxamine will bind to the PSA in the d-SPE, and thus are not recoverable. These compounds will need to be measured and quantified prior to this cleanup step. Figure 1. Sample preparation flowchart. 3 Results and discussion Table 1. GC/MS/MS Conditions. GC Conditions Sample preparation GC Agilent 7890B Using a simple extraction and SPE Column 1 Agilent HP-35MS, 15 m × 0.25 mm, 0.25 µm (p/n 122-3812) cleanup of the cannabis flower matrix Column 2 Agilent HP-5, 15 m × 0.25 mm, 0.25 µm (p/n 19091J-431) substantially reduces the issues Inlet Multimode (MMI) associated with QuEChERS extraction, Splitless, 4 mm single taper w/ deactivated fused silica wool Inlet liner which include spiking pH and exothermic Recommend: Agilent Ultra Inert (p/n 5190-2293) conditions that can degrade sensitive Column 1 flow 1.2 mL/min pesticides. In our approach, we eliminate Column 2 flow 1.25 mL/min the hydration step necessary with Inlet temperature 180 °C QuEChERS, thus increasing the recovery Inlet temperature program 180 to 280 °C at 400 °C/min of the more polar pesticides. Injection volume 2 µL 70 °C (1 minute) 35° C/min to 180 °C (0 minutes) For GC/MS/MS analysis, a cleanup Oven temperature program 10 °C/min to 200 °C (0 minutes) step was performed for each sample 8 °C/min to 300 (4.5 minutes) extract before analysis using the Run time 23.14 minutes Agilent Universal dispersive-SPE kit Column backflush Agilent Purged Ultimate Union (p/n G3186) (p/n 5982-0028). In this procedure, Intra-run, when deltamethrin elution passes the Ultimate Union 100 μL of the collected extract was Post run, 2.4 minutes at 4.0 mL/min added to 900 µL of hexane:acetone MS Conditions (1:1, v/v) in the 2-mL cleanup tube. The Spectrometer Agilent 7010 triple quadrupole GC/MS with high efficiency source (HES) slurry was vortexed and centrifuged, and Mode Electron ionization (EI) 300 µL of the supernatant was added Transfer line temperature 300 °C to 300 µL of hexane:acetone (1:1, v/v) Source temperature 230 °C (280 °C recommended) resulting in an overall 500‑fold dilution Quadrupole temperature 150 °C before analysis. For LC/MS/MS analysis, 50 µL of the Table 2. LC/MS/MS Conditions. collected extract was transferred to HPLC Conditions an autosampler vial containing 950 µL HPLC Agilent 1260 Infinity multisampler of acetonitrile, resulting in a 500‑fold Column Agilent InfinityLab Poroshell 120 Phenyl-Hexyl, 2.1 × 100 mm, 2.7 µm (p/n 695775-912) dilution. Column temperature 45 °C If additional sample cleanup is Injection volume 2 µL necessary due to the complexity of Mobile phase A 5 mM Ammonium formate + 0.1 % formic acid in 95:5 (v/v) water:methanol the coextracted matrix (see note in Mobile phase B 5 mM Ammonium formate + 0.1 % formic acid in 95:5 (v/v) methanol:water Figure 1), a third cleanup procedure Flow rate 0.4 mL/min can be followed using the Agilent MS Conditions dispersive-SPE kit (p/n 5982-5122).