AOAC CASP Cannabinoids ERP October Method Book

D. Preparation of Test Samples / Standard Solutions

Preparation of Standard Solutions a) Mobile phase A – To prepare 1 L of mobile phase A, weigh out 0.32 g of ammonium formate. Add 1 L of HPLC-grade water to yield a 5 mM solution. Add 1 mL formic acid. Invert and swirl to mix. Prepare weekly or as needed. b) Mobile phase B – To prepare 1 L of mobile phase B, pipette 1 mL formic acid into 1 L acetonitrile. Invert and swirl to mix. Prepare monthly or as needed. c) Sample diluent – To prepare 20 mL of Sample diluent, pipette 6 mL Mobile Phase A and 14 mL Mobile Phase B into a 20 mL glass vial. Prepared as needed d) Ibuprofen Check Standard Stock Solution – Prepare a 20 mg/mL stock solution of ISTD by dissolving 0.2 g of ibuprofen in 10 mL of acetonitrile. Accurately weigh ibuprofen and acetonitrile using an analytical balance. Vortex to mix. e) Ibuprofen Check Standard Working Solution – Pipette 5 µL of the 20 mg/mL Ibuprofen Check Standard Stock Solution into 95 µL of acetonitrile to form a 1 mg/mL solution. Prepare fresh for each batch. f) Phytocannabinoid Primary Stock Solution – Prepare a 40 µg/mL primary stock solution in a 2mL autosampler vial. Pipette 48 µL of Mobile Phase A, 8 µL of 1 mg/mL Ibuprofen Check Standard Working Solution, 16 µL of 0.5 mg/mL CBLA stock solution, and 8 µL of each 1 mg/mL phytocannabinoid standard (8 µL x 16). g) Phytocannabinoid Calibration Samples

Calibration Level (µg/mL) Vol (µL)/Standard Sample Diluent (µL)

25 125/Primary Stock Solution 75 10 50/Primary Stock Solution 150

2 40/Cal 10 160 0.5 50/Cal 2 150

0.1 40/Cal 0.5 160 0.05 80/Cal 0.1 80 0.02 80/Cal 0.05 120

h) Independent Check Standard (ICS) – Pipette 300 µL of Mobile Phase A, 605 µL of Mobile Phase B, 5 µL of Ibuprofen Check Standard Working Solution, 10 µL of 0.5 mg/mL CBLA stock solution, and 5 µL of each 1 mg/mL phytocannabinoid standard (5 µL x 16) into a 2 mL autosampler vial. Vortex to mix. D. Preparation of Test Samples a) Freeze chocolate samples at -80 °C for at least an hour. b) Immediately homogenize the entire sample. c) Accurately weigh 0.5 g of thoroughly comminuted subsample into a 15 mL centrifuge tube. d) Prepare a Method Blank with each sample batch by performing this procedure but omitting the addition of chocolate sample material. e) Add 10 mL of methanol to each sample, recording the accurate weight of solvent added. f) Add 50 µL of Ibuprofen Check Standard Stock Solution to each sample and vortex for 10 seconds. g) Place in an ultrasonic bath preheated to 50 °C and sonicate for 5 min. h) Agitate the sample manually to facilitate homogenization. i) Vortex for 10 seconds. j) Sonicate again at 50 °C for 5 minutes. k) Vortex for 10 seconds then cold stabilize samples at -20 °C for at least 2 hrs. l) Centrifuge at 3,000 rcf for 5 minutes. m) Pipette 10 µL of the resulting supernatant into 990 µL of Sample Diluent.