AOAC CASP Cannabinoids ERP October Method Book

AOAC Proposed Method SMPR 2017.019, Canopy Growth Corporation

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ii. DAD Conditions – Flow cell 3 mm; DAD wavelength 215 nm; DAD slit width 4 nm. iii. HDR-DAD Conditions – Two flow cells connected in series for increased linear range: (1) 60 mm (2) 3.7 mm. DAD wavelength 215 nm; DAD slit width 4 nm. iv. System Suitability – Monitor the performance of the HPLC system using a solution containing a mixture of cannabinoids. Passing system suitability parameters: standard deviation of replicate injections ≤ 1.83% for 5 injections; tailing factor at 5% peak height 0.8 – 1.5; column efficiency (plates) > 2000; capacity factor > 2; peak resolution > 1.5. Solvent blanks (i.e., methanol) must be

free of sample carry-over.

(b) Column – Agilent Poroshell 120 EC-C18; 4.6 x 250mm x 2.7µm, with guard column of

matching packing material; 4.6 x 5mm x 2.7µm.

(c) Bead bath – Maintaining 65°C ± 2°C.

(d) Ultrasonic water bath – With built-in timer for automatic shut-off. (e) Ultra low-temperature freezer – Maintaining -80°C ± 2°C.

(f) Centrifuge – Maintaining a speed of 1000 x g.

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 (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. the mark with HPLC-grade acetonitrile.

Fill to the mark with deionized water.

(f) Analytical Standards . – Certified Reference Materials; 1 mg/mL; Cerilliant Corporation.

i. d9-THC . Δ 9- tetrahydrocannabinol

ii. CBD. Cannabidiol

iii. d9-THCA. Δ 9-tetrahydrocannabinolic acid

iv. CBDA. Cannabidiolic acid

v. CBN. Cannabinol

vi. d8-THC. Δ 8- tetrahydrocannabinol vii. CBDVA . Cannabidivarinic Acid

viii. CBDV. Cannabidivarin ix. CBGA. Cannabigerolic acid

x. CBG. Cannabigerol xi. CBE. Cannabielsoin

xii. THCVA. Tetrahydrocannabivarinic acid

xiii. CBL. Cannabicyclol xiv. CBC. Cannabichromene

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