Materials
Certified reference material for THC, CBD, THCA-A, CBDA, CBN, THC-d3, CBD-d3, and CBN-d3 was purchased from Cerilliant Corporation (Round Rock, TX). All standards were analytical grade and were provided as either 1 mg/mL or 100 μg/mL (THC-d3, CBD-d3, CBN-d3) solution in methanol or acetonitrile. All solvents used were HPLC grade and purchased from Sigma-Aldrich (Missouri, USA).
Sample size and sampling strategy
Forty-five edible cannabis-infused products were used for this study. These items were collected over a 4-year period (2014–2018) through convenience sampling, that is, products were obtained either through a donation from budding entrepreneurs, confiscation from high school students, purchased on the black market at various events, or items for sampling at cannabis seminars. The details of the sample sources are as follows:
Source
|
Method of collection
|
Products collected
|
Year of collection
|
Legal status
|
---|
High schools
|
Submitted for testing
|
Baked goods
|
2014
|
Illegal
|
|
Submitted for testing
|
Candies
|
2015
|
Decriminalized
|
|
Submitted for testing
|
Baked goods and candies
|
2017
|
Decriminalized
|
Rastafarian
|
Submitted for testing
|
Baked goods and candies
|
2016
|
Decriminalized
|
Cannabis seminars
|
Donated
|
Baked Goods
|
2016
|
Decriminalized
|
|
Donated
|
Baked goods, candies, and preserves
|
2017
|
Decriminalized
|
Entertainment events
|
Bought by patrons
|
Baked goods
|
2016
|
Decriminalized
|
|
Bought by patrons
|
Baked goods and beverages
|
2018
|
Decriminalized
|
University students
|
Donated
|
Baked goods and candies
|
2016
|
Decriminalized
|
|
Donated
|
Baked goods and preserves
|
2018
|
Decriminalized
|
Cannabis companies
|
Donated
|
Baked goods and candies
|
2016
|
Decriminalized
|
|
Donated
|
Candies
|
2017
|
Decriminalized
|
University student vendor
|
Donated
|
Bread
|
2017
|
Decriminalized
|
Oregon dispensary
|
Purchased
|
Candies
|
2017
|
Decriminalized
|
|
Purchased
|
Chocolates
|
2017
|
Decriminalized
|
The sample size obtained and the non-probability sampling technique are a direct consequence of the fact that marijuana is illegal in Jamaica. In 2015, marijuana was decriminalized in Jamaica, and the majority of the items were obtained after this period.
Upon receipt of the products, they were assessed and placed into one of six categories namely baked goods, beverages, candies, chocolates, frozen foods, and preserves. The operational definitions guiding this classification process were as follows:
Baked Goods—cannabis-infused products made with cannabis butter or cannabis oil. They included brownies, bread, oatmeal cookies, chocolate chip cookies, carrot cake, fruit cake, coconut chocolate chip cookie, cupcakes, and Danish pastry.
Beverages—cannabis-infused drinks and included coffee and grape-flavored wine.
Cannabis candies—cannabis-infused candies and included gummy bears, chocolate candy, busta, kush candy, jewel candy, mango lollipop, tamarind ball, lime lollipop, and peanut cake, while chocolate bars were grouped separately.
Cannabis-infused ice cream and stewed June plum were categorized as frozen foods and preserves respectively.
Sample pre-treatment
Whole candies, cookies, brownies, and slices of cakes and bread were ground to a fine powder using a Proctor-Silex Coffee Grinder, while gummy candies and chocolates were cut into small pieces (approx. 0.5–2 mm). All samples were stored at − 20 °C until required for extraction.
Sample extraction
A modification of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) technique originally published by Wang et al. (2016) was used to extract cannabinoids from cannabis-containing foods (UCT, LLC 2015). The extracts were then diluted in preparation for instrumental analysis.
One gram of pretreated sample was weighed in a 50 mL silanized centrifuge tube. Ten milliliters of distilled water was then added and the mixture vortexed for 30 s. Ten milliliters of acetonitrile with 1% acetic acid was then added, and the mixture vortexed at medium speed for 1 min and shaken for an hour on a horizontal shaker at 150 rpm. Four grams of anhydrous MgSO4 + 1g NaCl (extraction salt) was added and the mixture vortexed for 1 min, centrifuged at 3000 rpm for 5 min, and the supernatant transferred to a silanized culture tube. The supernatant was then evaporated to dryness at 40 °C, under a gentle stream of nitrogen and the dried extract reconstituted in 1 mL of hexane to ethyl acetate (1:1). Serial dilutions of extracts were performed ranging from 50 to 400 times.
A volume of 100 μL of the diluted sample was removed to a silanized autosampler vial along with 50 μL of the working solution of internal standard (0.2 ppm) before drying at 40 °C under nitrogen. The dried extracts were derivatized by adding 100 μL MSTFA and heating at 70 °C for 30 min. One microliter of the derivatized extract was injected into the GC–MS system.
Preparation of standard solutions
A standard stock solution containing THC, CBD, CBN, CBDA, and THCA-A each at a concentration of 10 μg/mL was prepared in methanol. Working solutions at concentrations of 10, 100, and 1000 ng/mL were subsequently prepared by diluting the standard stock solution with methanol and stored at − 20 °C until needed for analysis. The internal standard working solutions (THC-d3, CBD-d3, CBN-d3) were prepared at a concentration of 200 ng/mL in methanol.
Calibrators containing THC, CBD, THCA-A, CBDA, and CBN at concentrations equivalent to 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, and 20 μg/g were prepared in duplicate in cannabis free brownie. The quality control sample consisted of a brownie containing 33.0 mg of THC. Calibrators and quality control samples were treated and processed in the same manner as test samples.
Instrumentation
GC-MS analysis was performed using an Agilent 7890 Gas Chromatograph equipped with a 7683 Series Autosampler and a 5975 Mass Selective Detector (MSD) using the Chemstation software. Analyte separation was achieved on a fused silica capillary column HP-5MS (30 m × 250 μm i.d. × 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The inlet temperature was set at 250 °C, and samples were injected in the splitless mode. The oven temperature was programmed at 80 °C (hold for 2 min) followed by an increase to 290 °C at a rate of 20 °C/min and held for 2 min. The total run time was 14.50 min with a solvent delay of 3 min.
The mass spectrometer was operated with the electron energy set at 70 eV. The retention times and characteristic mass fragments of the silyl derivatives of the cannabinoids were determined by recording the electron impact (EI) spectra in the total ion monitoring mode (scan range m/z 50–550). For quantitative analysis, the chosen characteristic mass fragments were monitored in the selected-ion-monitoring (SIM) mode: m/z 371,315, 386 for THC, m/z 390, 337, 301 for CBD, m/z 491,493, 492 for CBDA, m/z 487, 489, 488 for THCA, m/z 367, 368, 382 for CBN, m/z 374 for THC-d3, m/z 393 for CBD-d3, and m/z 370 for CBN-d3 (quantitative ions are in bold).
Method validation
Prior to application to samples, the method was validated for limits of detection and quantification, recovery, linearity, precision, and accuracy according to SWGTOX criteria (SWGTOX 2013).
Limits of detection and quantification (LOD and LOQ)
LOD and LOQ were determined in both candy and brownie matrices by using the standard deviation (S.D.) of the mean noise level over the retention time window of each analyte as follows: LOD = 3 × S. D and LOQ = 10 × S.D.
Recovery
Recovery samples were prepared by spiking blank brownie and candy samples with cannabinoid standards at 0.25 μg/g, 5 μg/g, and 20 μg/g. Using three replicates at each of the concentration levels, the absolute recoveries were calculated by comparing the peak areas of each cannabinoid obtained from spiked edible samples (brownie and candy) with those found after the direct injection of standard solutions at the same concentrations.
Linearity
Calibration curves were obtained from matrix-matched standard solutions at eight different concentrations for each cannabinoid from 0.1 μg/g to 20 μg/g.
Precision and accuracy
To test accuracy (recovery), spiked samples were prepared by adding three levels (low, medium, and high) of known concentrations of standards into three sets (n = 3) of replicates of blank edible samples (brownie and candy) giving a total of 12 samples per edible which were then extracted and quantified. For repeatability, samples were spiked similarly but with 6 sets (n = 6) of replicates and expressed as the relative S.D. (% RSD) of the calculated concentrations. Accuracy was expressed as the relative error of the calculated concentrations.
Statistical analysis
The descriptive statistics were calculated using the “Statistical Product and Service Solutions”—SPSS software. SPSS was also used to analyze the distribution of data with a box and whisker plot and the frequency of THC: CBD ratios with a histogram.