by David Egerton AL


A High-Throughput Method for Routine Analysis of Pesticide Residues on Cannabis


Cannabis sativa has long been used as a medicine, shamanic agent and euphoriant.1


Though medical legitimacy was regained in California in


1996 with the passage of Proposition 215, much of the Cannabis grown in the state has not been subjected to any level of quality testing. With the growth of the industry and the passage of a 2016 California referendum allowing access to Cannabis for all adults, the future cultivation and sale of Cannabis will be fully regulated starting in 2018. These regulations (still being drafted by the state) along with those already existing in several other states2


will likely include mandatory quality testing for both the


plant material and processed products. Of particular consumer interest and regulatory concern is the question of pesticide residues. Given the quasi-legal standing of many cultivators, the impetus to apply chemical pest controls has been unimpeded. Due to its federally illegal status, no pesticide is approved for use on Cannabis, and thus cultivators have had no guidance as to application limits.


In response to this challenge, many commercial laboratories have con- ducted their own research as to what products are in use and balance that with their analytical capabilities. This has led to a wide disparity in target lists available from different labs, as well as regulatory lists from different states. Although the market value of Cannabis exceeds that of most crops, batch sizes are quite small, leading to slim profit margins for nonretail businesses. Intense competition between Cannabis labs has further reduced pricing in an industry where testing is largely voluntary. There will be pressure on laboratories to keep testing fees low to accom- modate market demand.


Although Cannabis remains popular in its dry plant form, many other as- sociated forms exist. These include a wide variety of food and beverage products, as well as salves, tinctures, lotions, concentrated solvent- extracted oils and hashish. Each of these products provides for unique matrix challenges that can frustrate scientific analysis.


To address these needs, testing for pesticide residues in Cannabis must be sensitive, selective, fast and affordable. This article describes a robust, reliable method developed by CW Analytical (Oakland, Calif.) and AB Sciex (Toronto, Canada) that uses HPLC with triple-quadrupole mass spectrometry.


Materials and methods Sample preparation was conducted by homogenizing the starting


material and placing 500 mg of the sample into 10 mL of LC/MS-grade acetonitrile. The sample vial was then placed in a sonicator bath (Branson, Danbury, Conn.) for 10 minutes and placed on a shaker table for 1 min- ute. Supernatant was syringe-filtered through a 0.2-µm polyethersulfone (PES) filter and placed into an LC autosampler vial. Separation occurred on a Sciex ExionLC AC system employing a Phenomenex (Torrance, Calif.)


AMERICAN LABORATORY 32


Kinetex 5-µm biphenyl column (150 × 3.0 mm) and 5-mm guard column held at 40 °C. The aqueous phase (A) was 0.1% formic acid and 5 mM ammonium formate; organic phase (B) was 0.1% formic acid and 5 mM ammonium formate in 98/2 acetonitrile/water. A gradient of 10% B was held for 1 minute, followed by a ramp to 80% B at 4.30 minutes and 95% B at 8.7 minutes. This was held for 1.8 minutes before returning to starting conditions and holding for 3.5 minutes. The flow rate was 0.4 mL/min, and a 10-µL injection size was used. A Sciex 3500 triple-quadrupole system with Turbo V and electrospray ionization (ESI) probe with positive polarity was used for detection. Source temperature was set to 325 °C to minimize degradation of some compounds, curtain gas was set to 20, collision gas was 9 and ion-spray voltage was 5500. The ion source gases were both set to 60.


Designing the method to overcome matrix effects Cannabis and Cannabis-infused samples were prepared according to three methodologies: the method described above, the original unbuf- fered QuEChERS method, and the AOAC 2007.01 method. The extracts were each spiked at 1, 10, 40 and 80 ppb using a standard mix obtained from LGC Standards (Cumberland Foreside, Maine). To compare suppres- sion effects, analytes were quantified using a solvent-based calibration curve rather than a matrix-matched curve. Using these three methods, distinct matrices were prepared in triplicate: baked good, hard candy, gummi, glycerin tincture, wax, beverage, cow butter, beeswax-based balm, chocolate and Cannabis flower. For the purposes of this comparison, six pesticide residues popular among Cannabis growers were quantified: avermectin B1a, bifenazate, bifenthrin, dichlorvos, imidacloprid and myclobutanil. Results of the recovered residues are shown in Figure 1. The average recovery of all residues was 54.4% for the original method, 56.4% for the AOAC method and 89.5% for the dilute-and-shoot method described above. Based on these data, the researchers decided to proceed with a simple dilute-and-shoot method for sample preparation.


Further efforts to overcome ion suppression were made during method development. Each analyte began method development with a minimum of three applicable Q1tQ3 transitions. These transitions were then exam- ined in matrix samples, and quenched transitions were omitted from the final method. Changing to a biphenyl column chemistry and extending the gradient also aided in removing suppression zones from the target retention times. The full method contains a total of 47 pesticides of concern for Cannabis patients, one synergist (piperonyl butoxide), two plant-growth regulators (paclobutrazol and daminozide) and four afla- toxins (B1, B2, G1 and G2).


Although method development plays an integral role in establishing sensitivity of the method, routine maintenance and quality control (QC)


JANUARY/FEBRUARY 2017


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