As the number of U.S. states allowing the adult use of cannabis and cannabis products increases, so does the need for product testing before retail sale. States that have legalized recreational use have specified testing requirements for pesticide residues in cannabis flower and cannabis products. Because the specific pesticides and action levels vary from state to state, a comprehensive approach to residue analysis can meet the requirements of multiple U.S. state regulations with a single analysis. The challenge of quantifying pesticide residues in cannabis is complex because of the high concentration of cannabinoids and terpenes relative to the levels of pesticides that may be present. Here we present a straightforward acetonitrile extraction using a solid-phase extraction (SPE) cartridge and targeted dispersive solid-phase extraction (dSPE) cleanup. The final dilute extract is analyzed with both gas chromatography–tandem mass spectrometry (GC–MS/MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for a comprehensive target list (200+ compounds) that encompasses those identified on individual U.S. state lists. Limits of quantitation meet or exceed individual U.S. state requirements.
In our laboratory, sample preparation includes a serial extraction with acetonitrile followed by a solid-phase cleanup. A 1.0-g aliquot of homogenized sample was accurately weighed into a 50-mL centrifuge tube, with 15 mL of acetonitrile added to each tube along with a ceramic homogenizer. The tubes were sealed and mechanically shaken for 2 min at 1500 strokes/min. The acetonitrile was decanted through a conditioned Strata-X (Phenomenex) 33-µm solid-phase extraction (SPE) cartridge (500 mg/12 mL), and the eluent was collected in a second 50-mL centrifuge tube. The primary extraction tubes were rinsed with two additional 5-mL portions of acetonitrile, and decanted through the SPE cartridge. All eluent fractions were combined in the second 50-mL centrifuge tube. The solvent extract was brought to a final volume of 25 mL with acetonitrile. After this step we have a dilution factor of 25x from the initial sample amount.
As previously discussed, the analyses are conducted with both LC–MS/MS and GC–MS/MS systems. The GC–MS/MS system was an Agilent 7890B GC equipped with an Agilent 7010 MS/MS system with a high efficiency source (HES). The GC system was configured with a multimode inlet (MMI) and a Purged Ultimate Union (PUU) to allow for column backflushing. The MMI is capable of fast temperature ramping, allowing for a “cool” inlet injection. The initial inlet temperature was 180 °C. The temperature was ramped to 280 °C at 400 °C/min post-injection. Combining the “cool” inlet condition with a pulsed splitless injection helped reduce degradation of thermally labile compounds in the GC inlet. To improve peak shape for more-polar analytes, the PUU was placed at the midpoint of two analytical columns of different stationary phases (column 1 = HP-35MS, 15 m x 0.25 mm, 0.25-µm df; column 2 = HP-5, 15 m x 0.25 mm, 0.25-µm df). The backflush timing was configured to begin mid-run as determined by the retention time of the last compound exiting the PUU, and continued post-run to flush nonvolatile compounds from the injection port.
The LC–MS/MS system was an Agilent 1260 Infinity II with a Multisampler connected to an Agilent 6470 MS/MS system. A Poroshell 120 Phenyl-Hexyl column (100 mm x 2.1 mm, 2.7-µm dp) was selected because of its chromatographic performance and robustness. The LC column provided excellent chromatography at standard (non-ultrahigh-pressure liquid chromatography [UHPLC]) pump pressures, again reducing instrument downtime. A feature of the Multisampler is the ability to perform an injection pretreatment before sample injection. A 2-µL sample was sandwiched between two 10-µL aliquots of water, and injected onto the analytical column. This sample injection sandwich is not a dilution, it simply creates a polar environment for the sample injection, which improves peak shape for the early eluted compounds. Mobile-phase A was 5 mM ammonium formate with 0.1% formic acid in water–methanol (95:5) and mobile-phase B was 5 mM ammonium formate with 0.1% formic acid in methanol–water (95:5).
Both MS/MS systems share the same calibration technique. Calibration curves may use a linear fit (minimum five points) or a quadratic fit (minimum six points), with minimum correlation coefficients of 0.990 for all 215 compounds. The low calibrator concentration was 0.2 ng/mL for most compounds (92%), which corresponds to a sample concentration of 0.1 mg/kg after cleanup or final dilution factors. The upper range of the calibration curve was 10 ng/mL or 20 ng/mL depending on the individual analyte. All calibration curves used a 1/X weighting factor and exclude the origin. No internal standards were used, and all quantitative results were calculated using the external standard technique.
GC–MS/MS extracts were prepared for analysis using a combination of dSPE cleanup and dilution. For the dSPE cleanup, 100 µL of sample extract was added to a 2-mL disposable tube containing 50 mg of primary secondary amine (PSA), 50 mg of C18, 7.5 mg of graphitized carbon, 150 mg of magnesium sulfate, and 900 µL of 1:1 hexane–acetone. These tubes were capped, vortexed for 30 s, and centrifuged for 2 min. A 300 µL aliquot was removed from the tube and added to an autosampler vial containing 300 µL of hexane–acetone (1:1). At this point, the sample extract consists of 5% acetonitrile–95% hexane–acetone (1:1). This solvent system gives much better performance with GC compared to 100% acetonitrile. The combined dilutions from the cleanup step (10x) and post cleanup (2x), with the initial extraction (25x) result in an overall dilution factor of 500x. Using this approach, background noise and matrix in the final extract was significantly reduced (Figure 1) and recoveries for the target pesticides were within acceptance limits of 70–120%. (See upper right for Figure 1, click to enlage; Figure 1: Full-scan chromatograms: Full-scan data were collected for both GC–MS/MS [top] and LC–MS/MS [bottom]. The black chromatogram is the initial sample extract [post SPE column], the blue represents a 20x dilution of initial extract. The red chromatogram is post dSPE cleanup [50 mg PSA/50 mg C18/7.5 mg GCB/150 mg MgSO4].)
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Rick Jordan is the Laboratory Manager at Pacific Agricultural Laboratory in Sherwood, Oregon. Daniel Miller is the Technical Director at Pacific Agricultural Laboratory. Lilly Asanuma is a chemist at Pacific Agricultural Laboratory. Anthony Macherone is a Senior Scientist with Agilent Technologies and a visiting professor at The Johns Hopkins University School of Medicine. Direct correspondence to: [email protected]
How to Cite This Article
R. Jordan, L. Asanuma, D. Miller, and A. Macherone, Cannabis Science and Technology 1(2), 26-31 (2018).