AOAC CASP Cannabinoids ERP October Method Book

AOAC Proposed Method SMPR 2017.019

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Method for the Quantification of Cannabinoids in Edible Chocolate by High-Pressure Liquid

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Chromatography

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Method Authors: Alana Greaves, Jessica Gauthier, Ben Geiling

Canopy Growth Corporation, 1 Hershey Drive, Smiths Falls, ON, Canada, K7A 0A8 5 6 (Applicable for determination of 0.005 – 6.25 % w/w cannabinoids in dark, milk, and white chocolate. 7 For some cannabinoids, this range may be extended even further based on instrument linearity. This 8 method accurately quantifies d9-THC, CBD, d9-THCA, CBDA, CBN, and d8-THC, and can precisely monitor 9 an additional 9 cannabinoids. This method has been validated to include chocolate bars, chocolate chips, 10 and chocolate truffles, but does not include chocolates with nuts, fruit, or other ingredients which will 11 not fully homogenize upon melting. This method is suitable for HPLC-DAD as well as HPLC-HDR-DAD.) 12 A. Principle 13 Chocolate samples are melted and extracted with 90:10 methanol:chloroform. After a lipid- 14 removal step, filtered samples are analyzed by gradient reverse-phase high-pressure liquid 15 chromatography with a diode array detector. 16 B. Apparatus & Materials 17 (a) High-pressure liquid chromatograph (HPLC) – Liquid chromatograph with automated 18 sampler, quaternary gradient pump, de-gas module, column heater, and diode array 19 detector (DAD) or high dynamic range DAD (HDR-DAD). (Agilent Corporation, 5301 20 Stevens Creek Blvd., Santa Clara, CA, 95051. Tel: 1-800-227-9770)

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i. HPLC conditions – Column temperature 60°C; flow rate 1.5 mL/min; injection

volume 15.00 µL. Table 1 describes the mobile phase gradient.

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Table 1. HPLC mobile phase gradient.

HPLC Mobile Phase

Pump A Pump B Pump C Pump D

Deionized water

Methanol

Acetonitrile + 0.1% (v/v) phosphoric acid Deionized water + 0.1% (v/v) phosphoric acid

Time (min)

Pump A (%) 1

Pump B (%)

Pump C (%)

Pump D (%)

0.0 7.5

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0

66.0 66.0 66.0 66.0 66.0 66.0 66.0 66.0

34.0 34.0 19.0 19.0

10.5 14.0 15.0 21.0 22.0 25.0

15.0 15.0 34.0 34.0

0.0 0.0

0.0 0.0

34.0 34.0

1 Pump A is used for a seal wash.