7-27-2022_Draft-Agenda -ERP-CASPChemCont Methods

ERP Premeeting Method Review Book

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HPLC instrumentation as less sample matrix reaches the instrument. Column eluates are injected onto an HPLC with a fluorescent detector for quantitative analysis. Furthermore, electrochemical derivatisation using a KOBRA® CELL is employed for derivatisation of aflatoxin B1 and G1 to enhance fluorescence and to improve sensitivity of the method for these mycotoxins. In this study the immunoaffinity columns were only used for detection of aflatoxins and ochratoxin A since these were the only toxins that were legislated for in cannabis and in cannabis derived products, however the method could be extended in the future should legislation for other mycotoxins be introduced. B. Apparatus & Materials 1. Samples.—A- Cannabis flower; B- vape liquid consisting of cannabis distillate diluted with terpenes; C- refined resin (distillate); D- cannabis isolate, ca. 99% w/w CBD, E- edible oil, strain A, cannabis distillate dissolved in medium chain triglyceride (MCT) oil; F- edible oil, strain B, cannabis distillate dissolved in MCT oil; G- edible oil, strain C, cannabis distillate dissolved in MCT oil; H – cannabis dark chocolate (70% cocoa); I - cannabis milk chocolate (50% cocoa); J- pectin-based cannabis gummies; K-cannabis non-alcoholic flavored beverage; Products B-K were derived from extractions from cannabis flowers (i.e., no synthetic cannabinoids used). 2. AO ZON PREP® multi-mycotoxin IACs in a 3ml, wide-body format containing monoclonal antibodies specific to both aflatoxins (AFT) and ochratoxin A (OTA), obtained from R-Biopharm Rhone Ltd (Glasgow, UK). The columns have a capacity of 150 ng of total aflatoxins and 200 ng of OTA. Recoveries were not less than 80% for aflatoxins B1, B2, G1 and G2 when 5 ng of equimolar aflatoxins B1, B2, G1 and G2 was applied in 10 mL of methanol–PBS (10:90) and not less than 85% for OTA when applied as a standard solution in methanol–PBS (10:90) containing 5 ng of OTA. 3. Distilled/deionized water (suitable for use with LC) obtained from an in-lab water purifier (Milli-Q). LC-grade methanol, LC-grade acetonitrile, nitric acid, and potassium bromide from Fisher Scientific (Ottawa, Ontario, Canada). 4. Tween 20 from Sigma-Aldrich Ltd (Oakville, Ontario, Canada). 5. Phosphate buffered saline (PBS) tablets from VWR International (Mississauga, Ontario, Canada). 6. Standards.— Aflatoxin standard solution (CRM46304) containing 1 μg/mL aflatoxin B1, 0.3 μg/mL aflatoxin B2, 1 μg/mL aflatoxin G1, and 0.3 μg/mL aflatoxin G2 in methanol was obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Ochratoxin A standard solutions (10.0 μg/mL and 0.98 μg/mL in methanol) were obtained from Trilogy (Washington, MO 63090 USA) 7. (a) LC with fluorescence detection.—Agilent 1260 Infinity II LC system with quaternary pump and G7121B fluorescent detector (Agilent Technologies Canada Inc. Mississauga, ON Canada). The system was connected to a post-column KOBRA® CELL–K01 (R-Biopharm Rhone Ltd, Glasgow, UK). 7. (b) LC.—An Inertsil ODS- 3V (150 × 4.6mm, 5 μm particle size) analytical column with an Intersil ODS-3 (10 × 4.0 mm, 5 μm particle size) guard cartridge was used for the LC separation (GL Sciences, Peterborough, ON, Canada). 7. (c) The mobile phase A is isocratic for 6.5 min at a flow rate of 1.6 mL/min, then switching linearly over a period of 1 min to 30% mobile phase A and 70% mobile phase B at a flow rate of 2.0 mL/min (total run time of 7.5 min), and held under these conditions for a further 4.5 min (total run time of 12.0 min) before switching linearly back to starting conditions by 13.0 min, and holding these conditions for 2 min (total run time of 15.0 min). The injection volume was 100 μ L, and the column temperature was 40°C. The detector had excitation and emission wavelengths of 365 nm and 442nm respectively for 0 to 8.5 min for aflatoxin analysis then

July 2022