AOAC CASP Cannabinoids ERP October Method Book

C. Reagents

(a) Extraction Solution. – 90:10 methanol:chloroform solution. Add 100 mL of HPLC-grade chloroform to a 1L volumetric flask. Fill to the mark with HPLC- grade methanol. (b) Mobile Phase A. – Deionized water. (c) Mobile Phase B. – HPLC-grade methanol. (d) Mobile Phase C. - Acetonitrile + 0.1% (v/v) phosphoric acid. 1.175 mL of phosphoric acid (85% v/v), or equivalent is added to a 1L volumetric flask using a verified pipette. Fill to the mark with HPLC-grade acetonitrile. (e) Mobile Phase D. – Deionized water + 0.1% (v/v) phosphoric acid. 1.175 mL of phosphoric acid (85% v/v), or equivalent is added to a 1L volumetric flask using a verified pipette. Fill to the mark with deionized water. (f) Analytical Standards. – Certified Reference Materials; 1 mg/mL; Cerilliant Corporation. i. d9-THC. Δ9- tetrahydrocannabinol ii. CBD. Cannabidiol iii. d9-THCA. Δ9-tetrahydrocannabinolic acid iv. CBDA. Cannabidiolic acid v. CBN. Cannabinol x. CBG. Cannabigerol xi. CBE. Cannabielsoin xii. THCVA. Tetrahydrocannabivarinic acid xiii. CBL. Cannabicyclol xiv. CBC. Cannabichromene xv. CBCA. Cannabichromenic Acid (g) Analytical Standards. – Certified Reference Materials; 2.5 mg; Toronto (a) Preparation of calibration curve. – Prepare a calibration curve in methanol from certified analytical standards for each cannabinoid of interest. Calibration curves may be prepared individually, or mixed. The linear range of each compound’s prepared calibration curve should range from 0.1% w/v to 0.00001% w/v. Points at either extremity of the curve which reduce the curve’s linearity (R2 < 0.99), or whose signal-to-noise ratio is less than 10 should not be used. All calibration curve solutions should be prepared using a gas-tight syringe using methanol as a diluent. Calibration curves are plotted as Peak Area vs. Concentration (% w/v), with the x-intercept set to 0.0 and the line of best fit following the equation Peak area = (Slope)(Concentration). The instrument’s minimum linear range must be equal to 0.6 – 400 ppm to accurately capture cannabinoid concentrations of 0.008 – 5% w/w in chocolate samples. (b) Preparation of test samples. i. For solid chocolate, including chocolate bars and chocolate chips – Homogenize the solid chocolate sample using a mortar and pestle. Accurately weigh 0.08000 g of chocolate (with a tolerance of +5%) into a glass test tube and record the mass. ii. For chocolate truffles – Place the entire truffle into a closed container, such as a 50 mL centrifuge tube. Place in a 65°C bead bath until the chocolate is melted, about 10 minutes. Using a lab spatula or stir bar, thoroughly mix the melted chocolate. Accurately weigh 0.08000 g of chocolate (with a tolerance of +5%) into a glass test tube and record the mass. Note: This method has been validated for chocolate truffles which can fully homogenize upon melting, such as those dusted with cocoa powder or coated with solid chocolate. This method does not apply to chocolate truffles with nuts, fruit, or other ingredients which will not fully homogenize when melted. Research Chemicals i. CBE. Cannabielsoin vi. d8-THC. Δ8-tetrahydrocannabinol vii. CBDVA. Cannabidivarinic Acid viii. CBDV. Cannabidivarin ix. CBGA. Cannabigerolic acid

D. Preparation of Test Samples / Standard Solutions