AOAC CASP Cannabinoids ERP October Method Book

High-pressure liquid chromatography (HPLC) coupled to an ultra-violet (UV) detector offers excellent 30 separation of major and minor cannabinoids. More specifically, the use of a diode array detector (DAD) 31 allows detection at many wavelengths simultaneously. As a result, spectral libraries can be created for 32 analytes and matched against signal peaks in samples to confirm analyte identity and ensure adequate 33 peak purity (i.e., the lack of interfering peaks). Finally, by stacking two DAD units in tandem with different 34 flow cell path lengths (i.e., high dynamic range DAD), the linear range of the instrument is further 35 extended and able to capture a wider range of concentrations in a single sample dilution. The present 36 method is suitable to be run by HPLC using either a single DAD detector, or an HDR-DAD detector. The 37 data presented in this study were collected using an HDR-DAD detector. 38 The present method allows for the quantification of 15 cannabinoids, including cannabielsoin (CBE), the 39 major oxidative product of CBD. Validation encompassed 6 of these cannabinoids, Δ 9-THC, Δ 8-THC, CBD, 40 CBN, Δ 9-THCA, and CBDA, in multiple chocolate materials: dark chocolate bars, milk chocolate bars, white 41 chocolate chips, and dark chocolate truffles. The instrument’s response of these cannabinoids was highly 42 linear, with analytical ranges spanning 3 orders of magnitude, which allowed a simple sample preparation 43 method with a resulting single injection on HPLC. 44

METHOD

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TEST AND NEGATIVE CONTROL MATERIALS

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(a) Negative Control Materials – Chocolates of various types and formats were used in this study as

negative controls and to create test materials.

(1) Dark chocolate (Lindt, 70% cocoa solids). Chocolate bar

(2) Milk chocolate (No Name). Chocolate bar (3) White chocolate (Hershey’s). Chocolate chips

(4) Dark chocolate (No Name). Truffle (Made in-house). Truffles were made by mixing 8 oz dark chocolate together with ½ cup heavy whipping cream and 1 tsp vanilla extract, rolled

into 9-12 g balls and rolled in cocoa powder.

(b) Test Materials – Test materials used in the validation study were chosen to represent the analytical reported range for each compound in the method. Accuracy was performed by spiking certified reference materials into cannabis-free chocolate. Precision, specificity, and stability were

performed by replicate testing of in-house reference materials.

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