AOAC CASP Cannabinoids ERP October Method Book

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sample for 30 minutes using an ultrasonic water bath, followed by vortexing for 30 seconds. Place the sample in the ultra low-temperature freezer (-80°C) for 15 minutes to ensure complete solidification of lipids. Remove the sample from the freezer and immediately centrifuge at 1000 x g for 3 minutes. Filter the centrifuged solution into an HPLC vial using a glass or plastic syringe and a 0.2 µm PTFE syringe filter, discarding the first few milliliters of filtrate in case cannabinoids bind

to the membrane. Analyze the sample by HPLC.

CHROMATOGRAPHY

(a) HPLC conditions – Column temperature 60°C; flow rate 1.5 mL/min; injection volume 15.00 µL; DAD wavelength 215 nm; DAD slit width 4 nm. For DAD detector, use 3 mm flow cell. For HDR- DAD detector, 2 flow cells are connected in series: (1) 60 mm flow cell, (2) 3.7 mm flow cell. Table (b) 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 2 describes the mobile phase gradient.

free of sample carry-over.

(c) Retention Times – Figure 1 shows the separation of the 15 cannabinoids in this method.

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CALCULATIONS

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(a) Use the same integration parameters for the test solutions as for the calibration curve solutions by choosing the same peak width, threshold settings, and other integration parameters. Use peak area for quantification. Calculate the concentration of each cannabinoid in the test sample using

the slope of the calibration curve and the following equation:

10 mL Test sample mass (g)

Peak Area in test sample Slope ×

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Conc. in test sample (% w/w) =

172

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PROCEDURES USED FOR VALIDATION

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(a) Specificity – The resolution of the 6 validated cannabinoids was determined by replicate injection (n = 6) of a master mix of the validated and non-validated cannabinoid analytical standards. Acceptable separation was set to a resolution of ≥ 1.5. The spectra of the analytical standards were

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