AOAC CASP Cannabinoids ERP October Method Book

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AOAC SMPR ® 2017.019

Reproducibility.— The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility

relative standard deviation (%RSD R ). 5 Method Performance Requirements See Tables 4 and 5.

Standard Method Performance Requirements (SMPRs®) for Quantitation of Cannabinoids in Edible Chocolate

6 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. 7 Reference Material(s) See Tables 1 and 2 for examples of acceptable reference materials. Also see: AOAC Technical Division on Reference Materials (TDRM) Databaseathttp://www.aoac.org/aoac_prod_imis/AOAC_Member/ MEMCF/TDCF/TDRMDbInfo.aspx Restek Corp. Restek® Reference Standards at http://www. restek.com/Reference-Standards Shimadzu Corp. Shimadzu Certified Reference Materials at http://www.ssi.shimadzu.com/cannabis_stds Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F : Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma.aoac.org/app_f.pdf ) 8 Validation Guidance See “Clarification on Testing Materials for Cannabinoid SMPRs” (http://www.aoac.org/AOAC_Prod_Imis/AOAC_Docs/SPSFAM/ ValidationGuidanceClarificationforSMPR2017_001and_002.pdf ) Method performance does not require demonstration of matrix homogeneity; the chocolate is viewed as the vehicle for cannabinoid delivery. Method performance should be demonstrated using a representative subsample of the matrix which assumes uniform distribution of cannabinoids. Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma.aoac. org/app_d.pdf) Appendix F : Guidelines for Standard Method Performance Requirements , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma.aoac.org/app_f.pdf) AppendixK : Guidelines for Dietary Supplements and Botanicals , Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http:// www.eoma.aoac.org/app_k.pdf ) 9 Maximum Time-to-Result None Approved by the AOAC Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM) on September 24, 2017. Final Version Date: November 13, 2017.

Intended Use: Consensus-Based Reference Method for Use by Trained Technicians in a Laboratory for Routine Quality Assurance Testing 1 Purpose AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC stakeholder panels composed of representatives from industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for method being considered for Performance Tested Methods SM or AOAC Official Methods of Analysis SM , and can be used as acceptance criteria for verification at user laboratories. 2 Applicability Identification and quantification of individual cannabinoids (as listed in Tables 1 and 2) in finished edible chocolate as listed in Table 3. 3 Analytical Technique Any analytical technique(s) that measures the analytes of interest and meets the following method performance requirements is/are acceptable. 4 Definitions Chocolate. — Any edible solid confection substantially consisting of “chocolate” (i.e., dark, milk, or white) without added inclusions. Limit of quantitation (LOQ). — Minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Quantitative method.— Method of analysis which response is the amount of the analyte measured either directly (enumeration in a mass or a volume) or indirectly (color, absorbance, impedance, etc.) in a certain amount of sample. Recovery.— The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. Repeatability.— Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ).

Table 1. Required cannabinoids Common name

Abbreviation

IUPAC name

CAS No.

Molecular structure Reference material

Cannabidiol

CBD

2-[(1 R ,6 R )-6-isopropenyl-3- methylcyclohex-2-en-1-yl]-5- pentylbenzene-1,3-diol

13956-29-1

Restek Cerilliant

Sigma-Aldrich API Standards Echo Pharm Lipomed AG

Cannabidiolic acid

CBDA 2,4-Dihydroxy-3-[(1 R ,6 R )-3-methyl- 6-prop-1-en-2-ylcyclohex-2-en-1-yl]- 6-pentylbenzoic acid

1244-58-2

Cerilliant USP Restek Lipomed AG Echo Pharmaceutical

Cannabinol

CBN 6,6,9-Trimethyl-3-pentyl-benzo[ c ] chromen-1-ol

521-35-7

Cerilliant Restek

Δ9 Tetrahydro-cannabinol

THC

(−)-(6a R ,10a R )-6,6,9-Trimethyl-3- pentyl-6a,7,8,10a-tetrahydro-6 H - benzo[ c ]chromen-1-ol

1972-08-3

Cerilliant USP Echo Pharmaceuticals

Tetrahydro-cannabinolic acid

THCA

(6a R ,10a R )-1-hydroxy-6,6,9- trimethyl-3-pentyl-6a,7,8,10a- tetrahydro-6 H -benzo[ c ]chromene-2- carboxylic acid

23978-85-0

Cerilliant USP Echo Pharmaceuticals

Table 2. Additional, desirable cannabinoids Name Abbreviation

IUPAC name

CAS No.

Molecular structure

Reference material Cerilliant Sigma-Aldrich Echo Pharmaceuticals

Cannabichromene

CBC 2-Methyl-2-(4-methylpent-3- enyl)-7-pentyl-5-chromenol

20675-51-8

Cannabichromenicacid CBCA 5-Hydroxy-2-methyl-2-(4-methyl- 3-penten-1-yl)-7-pentyl-2H- chromene-6-carboxylic acid

20408-52-0

Cerilliant

Cannabidivarinic acid

CBDVA 2,4-Dihydroxy-3-[(1 R ,6 R )-3- methyl-6-prop-1-en-2-ylcyclohex- 2-en-1-yl]-6-propylbenzoic acid

31932-13-5

Cerilliant

Cannabigerol

CBG 2-[(2 E )-3,7-dimethylocta-2,6- dienyl]-5-pentyl-benzene-1,3-diol NIST: 1,3-Benzenediol, 2-(3,7-dimethyl-2,6-octadienyl)- 5-pentyl-

25654-31-3

Cerilliant Lipomed AG Echo Pharmaceuticals SPEX Certiprep Tocris (UK) Cerilliant Echo Pharmaceuticals SPEX Certiprep

NIST: 2808-33-5

Cannabigerolic acid

CBGA

3-[(2 E )-3,7-dimethylocta- 2,6-dienyl]-2,4-dihydroxy-6- pentylbenzoic acid

25555-57-1

Cannabidivarin

CBDV 2-((1 S ,6 S )-3-methyl-6-(prop-1- en-2-yl) cyclohex-2-enyl)-5- propylbenzene-1,3-diol

24274-48-4

Cerilliant SPEX Certiprep

Δ8 Tetrahydro-cannabinol

Δ8 THC 6,6,9-Trimethyl-3-pentyl- 6a,7,10,10a-tetrahydrobenzo[ c ] chromen-1-ol

5957-75-5

Cerilliant SPEX Certiprep

Tetrahydro-cannabivarin THCV

6,6,9-Trimethyl-3-propyl- 6a,7,8,10a-tetrahydro-6 H - benzo[ c ]chromen-1-ol

28172-17-0

Cerilliant USP

Tetrahydrocannabivarin acid

THCVA

28172-17-0

Cerilliant

Table 3. Matrices Chocolate chips Chocolate bars Chocolate truffles White chocolate

Table 5. Method performance requirements (part 2) for individual cannabinoids a

Range (% by weight)

≤0.008–1

Parameter

Recovery, %

90–110

95–105

RSD r , % RSD R , %

≤5 ≤8

≤4 ≤5

Milk chocolate Dark chocolate

a Analytical range based on following assumptions: Smallest amount of cannabinoid in a serving: 10 mg Largest amount of cannabinoid in a serving: 100 mg

Table 4. Method performance requirements (part 1) for individual cannabinoids Parameter Requirement Limit of quantitation (LOQ) (% by weight) ≤0.008 Minimum analytical range (% by weight) 0.008–5 a a Lower concentrations may be acceptable as applicable for cannabinoids listed in Table 2.

Smallest serving size: 2.5 g Largest serving size: 120 g

Lowest concentration: 10 mg/120 g = 0.008% Highest concentration: 100 mg/2.5 g = 4%

AOAC Method Submission Form

Submission Date

2020-07-08 12:08:21

Submitter Name

Matthew Noestheden

Submitter Email

matt@suprarnd.ca

Organization

Supra Research and Development

Method Type

Cannabis

Resubmission?

Method Name

Quantitation of Cannabinoids in Edible Chocolate

Method Author(s)

James, W. Favell, Ryan Hayward, Seamus Riordan-Short, Nahanni Sagar, Rob O'Brien, Matthew Noestheden

Method Applicability

Applicable for the quantitation of 17 phytocannabinoids in edible chocolates [white, dark and milk] from 0.008 – 4 % w/w.

A. Principle

Phytocannabinoids are extracted from white, milk or dark chocolates by adding methanol and heating the resulting solution to 50 °C with ultrasonic-assisted extraction. Extracts are cold stabilized for two hours prior to analysis. Analyte separation is achieved by liquid chromatography, followed by detection using a photodiode array. Other detectors are viable alternatives, provided they meet the standards set out in SMPR2017_019. Equivalent apparatus, reagents and materials may be substituted. Apparatus a) Micropipettes — adjustable volume, with maximum capacities of 20 µL, 200 µL, 1000 µL and 5000 µL b) Analytical balance — should be capable of achieving 0.2 mg linearity, 0.1 mg readability and 0.1 mg repeatability c) Top loading balance — should be capable of achieving 0.02 g linearity, 0.01 g readability and 0.01 g repeatability d) Centrifuge — must be able to reach 3,000 rcf with 15 mL centrifuge tubes e) uHPLC system — the pumping system should possess a pressure limit of at least 1,000 bar with a flow accuracy of at least 0.1%, a thermostated sample compartment and column oven and a detector capable of meeting the method performance criteria outlined herein. f) HPLC column — must be able to resolve 17 phytocannabinoids and ibuprofen in the specified matrices (resolution could be strictly via chromatography, or in tandem with the detector [e.g., with a mass spectrometer, where chromatographic resolution of all target analytes is not required if they are mass resolved]). g) Software — should have data acquisition and processing capabilities, including auto and manual integration functions h) Heated ultrasonic bath — must reach a stable temperature of 50 ± 5 °C i) Vortex mixer Materials a) 2 mL Amber borosilicate glass autosampler vials with PTFE-lined caps b) Polypropylene centrifuge tubes (15 mL) c) 4 mL borosilicate glass vials with PTFE-lined caps d) Pasteur pipettes

B. Apparatus & Materials

C. Reagents

Reagents All solvents were HPLC-grade or better. All phytocannabinoids were purchased from Cerilliant Corp. and used as received (Round Rock, TX). a) Acetonitrile, Methanol, Water – Fisher Scientific (Hampton, NH) b) Formic acid – Reagent Grade, ≥95% (Honeywell Laboratory Solutions, Charlotte, NC) c) Ammonium formate – Sigma-Aldrich (St. Louis, MO) d) Cannabinchromene (CBC) – 1.0 mg/mL in methanol e) Cannabinchromenic acid (CBCA) – 1.0 mg/mL in acetonitrile f) Cannabidiol (CBD) – 1.0 mg/mL in methanol g) Cannabidiolic acid (CBDA) – 1.0 mg/mL in acetonitrile h) Cannabidivarin (CBDV) – 1.0 mg/mL in methanol i) Cannabidivarinic acid (CBDA) – 1.0 mg/mL in acetonitrile j) Cannabigerol (CBG) – 1.0 mg/mL in methanol k) Cannabigerolic acid (CBGA) – 1.0 mg/mL in acetonitrile n) Δ8-Tetrahydrocannabinol (Δ8-THC) – 1.0 mg/mL in methanol o) Δ9-Tetrahydrocannabinol (Δ9-THC) – 1.0 mg/mL in methanol p) Δ9-Tetrahydrocannabinolic acid (THCA) – 1.0 mg/mL in acetonitrile q) Tetrahydrocannabivarin (THCV) – 1.0 mg/mL in methanol r) Tetrahydrocannabivarinic acid (THCVA) – 1.0 mg/mL in acetonitrile s) Cannabicyclol (CBL) – 1.0 mg/mL in acetonitrile l) Cannabinol (CBN) – 1.0 mg/mL in methanol m) Ibuprofen (Sigma-Aldrich, St. Louis, MO)

t) Cannabicyclolic Acid (CBLA) – 0.1 mg/mL in methanol u) Cannabinolic Acid (CBNA) – 1.0 mg/mL in acetonitrile

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.

E. Determinations

All blanks, calibrators, unknowns and quality control samples are analyzed using the same method.

Column Ascentis Express 90 Å C18 15 cm x 2.1 mm x 2 µm Column Temp 30 °C Mobile Phase A 5 mM ammonium formate in water + 0.1% v/v formic acid Mobile Phase B 0.1% formic acid in acetonitrile Autosampler Compartment 8 °C Flow Rate 0.4 mL/min Gradient 0.0 min: 70% B

3.0 min: 90% B 5.0 min: 90% B 5.1 min: 98% B 6.0 min: 98% B 6.1 min: 70% B 8.0 min: 70% B Detection 228 nm Injection Volume 25 µL

Elution Order Ibuprofen, CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, Δ9-THC, Δ8-THC, CBL, CBC, THCA, CBCA, CBLA

H. Calculations

a) Phytocannabinoid concentrations (% w/w) are calculated using the calibration parameters generated by the processing software with each sample batch analyzed. b) Sample and solvent masses are corrected as necessary. For example, the actual mass of sample or methanol used for extraction divided by the nominal amount – either 0.5 g of sample or 7.91 g for 10 mL methanol at 25 °C, respectively. c) Expected concentrations of cannabinoids and ibuprofen are outlined below (na – not applicable):

Sample type Phytocannabinoid (µg/mL) Ibuprofen (µg/mL) Unknown unknown 1 Method Blank na 1 ICS 5 5 RRS incurred + 1 2

Reference Materials Used:

a) No reference materials were available to support this work. b) Notwithstanding this limitation, the milk chocolate used was a rejected lot from a commercial manufacturer – it contains THC and CBD. Using procedures compliant with ISO/IEC 17034, this material was assessed for homogeneity and values were assigned for THC and CBD accordingly. These values were used as a reference point to demonstrate full method applicability for neutral phytocannabinoids.

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SMPR2017_019_ChocolateCannabinoids_SupraRnD.docx

Author Notes / Comments

The attached manuscript contains complete method performance details, as well as details regarding the study design for the single-laboratory validation.

Quantitating Cannabinoids in Edible Chocolates

In support of SMPR2017_019 Quantitating Cannabinoids in Edible Chocolates Liquid Chromatographic Separation with Photodiode Array Detection (Applicable for the quantitation of 17 phytocannabinoids in edible chocolates [white, dark and milk] from 0.008 – 4 % w/w) James W. Favell 1,2 , Ryan Hayward 1 , Seamus Riordan-Short 1 , Nahanni Sagar 1 , Rob O’Brien 1,2 , and Matthew Noestheden 1,2 * 1 Supra Research and Development, 106-2293 Leckie Rd. Kelowna, BC, V1X 6Y5 2 Department of Chemistry, I.K. Barber School of Arts and Sciences, University of British Columbia, Charles E. Fipke Centre for Innovative Research, 3247 University Way, Kelowna, BC V1V 1V7 * Corresponding author See Tables 1-3 for the results of the single laboratory validation in milk, white and dark chocolate that support the acceptance of the proposed method with respect to SMPR2017_019. A. Principle Phytocannabinoids are extracted from white, milk or dark chocolates by adding methanol and heating the resulting solution to 50 °C with ultrasonic-assisted extraction. Extracts are cold stabilized for two hours prior to analysis. Analyte separation is achieved by liquid chromatography, followed by detection using a photodiode array. Other detectors are viable alternatives, provided they meet the standards set out in SMPR2017_019. B. Apparatus, Reagents & Materials Equivalent apparatus, reagents and materials may be substituted. Apparatus a) Micropipettes — adjustable volume, with maximum capacities of 20 μL, 200 μL, 1000 μL and 5000 μL b) Analytical balance — should be capable of achieving 0.2 mg linearity, 0.1 mg readability and 0.1 mg repeatability c) Top loading balance — should be capable of achieving 0.02 g linearity, 0.01 g readability and 0.01 g repeatability d) Centrifuge — must be able to reach 3,000 rcf with 15 mL centrifuge tubes e) uHPLC system — the pumping system should possess a pressure limit of at least 1,000 bar with a flow accuracy of at least 0.1%, a thermostated sample compartment and column oven and a detector capable of meeting the method performance criteria outlined herein. f) HPLC column — must be able to resolve 17 phytocannabinoids and ibuprofen in the specified matrices (resolution could be strictly via chromatography, or in tandem with the detector [e.g., with a mass spectrometer, where chromatographic resolution of all target analytes is not required if they are mass resolved]). g) Software — should have data acquisition and processing capabilities, including auto and manual integration functions h) Heated ultrasonic bath — must reach a stable temperature of 50 ± 5 °C

Quantitating Cannabinoids in Edible Chocolates

i) Vortex mixer Reagents All solvents were HPLC-grade or better. All phytocannabinoids were purchased from Cerilliant Corp. and used as received (Round Rock, TX). a) Acetonitrile, Methanol, Water – Fisher Scientific (Hampton, NH) b) Formic acid – Reagent Grade, ≥95% (Honeywell Laboratory Solutions, Charlotte, NC)

c) Ammonium formate – Sigma-Aldrich (St. Louis, MO) d) Cannabinchromene (CBC) – 1.0 mg/mL in methanol e) Cannabinchromenic acid (CBCA) – 1.0 mg/mL in acetonitrile f) Cannabidiol (CBD) – 1.0 mg/mL in methanol g) Cannabidiolic acid (CBDA) – 1.0 mg/mL in acetonitrile h) Cannabidivarin (CBDV) – 1.0 mg/mL in methanol i) Cannabidivarinic acid (CBDA) – 1.0 mg/mL in acetonitrile j) Cannabigerol (CBG) – 1.0 mg/mL in methanol k) Cannabigerolic acid (CBGA) – 1.0 mg/mL in acetonitrile n) Δ8 -T etrahydrocannabinol (Δ8 -THC) – 1.0 mg/mL in methanol o) Δ9 -T etrahydrocannabinol (Δ9 -THC) – 1.0 mg/mL in methanol p) Δ9 -Tetrahydrocannabinolic acid (THCA) – 1.0 mg/mL in acetonitrile q) Tetrahydrocannabivarin (THCV) – 1.0 mg/mL in methanol r) Tetrahydrocannabivarinic acid (THCVA) – 1.0 mg/mL in acetonitrile s) Cannabicyclol (CBL) – 1.0 mg/mL in acetonitrile t) Cannabicyclolic Acid (CBLA) – 0.1 mg/mL in methanol u) Cannabinolic Acid (CBNA) – 1.0 mg/mL in acetonitrile Materials a) 2 mL Amber borosilicate glass autosampler vials with PTFE-lined caps b) Polypropylene centrifuge tubes (15 mL) c) 4 mL borosilicate glass vials with PTFE-lined caps d) Pasteur pipettes C. Preparation of Standard Solutions l) Cannabinol (CBN) – 1.0 mg/mL in methanol m) Ibuprofen (Sigma-Aldrich, St. Louis, MO)

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.

Quantitating Cannabinoids in Edible Chocolates

e) Ibuprofen Check Standard Working Solution – Pipette 5 µ L of the 20 mg/mL Ibuprofen Check Standard Stock Solution into 95 µ L of acetonitrile to form a 1 mg/mL solution. Prepare fresh for each batch. f) Phytocannabinoid Primary Stock Solution – Prepare a 40 µ g/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 µ L of each 1 mg/mL phytocannabinoid standard (8 µ L x 16). g) Phytocannabinoid Calibration Samples

Calibration Level (µg/mL)

Vol (µL)/Standard

Sample Diluent (µL)

125/Primary Stock Solution

50/Primary Stock Solution

150

40/Cal 10

160

0.5

50/Cal 2

150

0.1

40/Cal 0.5

160

0.05

80/Cal 0.1

0.02

80/Cal 0.05

120

Quantitating Cannabinoids in Edible Chocolates

n) Vortex for 10 s. o) Prepare an ICS, Routine Recovery Sample (RRS) and RRS Duplicate with each sample batch. p) To prepare an RRS, pipette 193 µL Sample Diluent, 5 µL of Phytocannabinoid Primary Stock Solution and 2 µL of a sample supernatant into a 2 mL autosampler vial with insert. Vortex to mix. q) To prepare an RRS Duplicate, repeat the previous step with the same supernatant. E. Determinations All blanks, calibrators, unknowns and quality control samples are analyzed using the same method.

Column

Ascentis Express 90 Å C18 15 cm x 2.1 mm x 2 µm

Column Temp

30 °C

Mobile Phase A

5 mM ammonium formate in water + 0.1% v/v formic acid

Mobile Phase B

0.1% formic acid in acetonitrile

Autosampler Compartment 8 °C Flow Rate

0.4 mL/min

Gradient

0.0 min: 70% B 3.0 min: 90% B 5.0 min: 90% B 5.1 min: 98% B 6.0 min: 98% B 6.1 min: 70% B 8.0 min: 70% B

Detection

228 nm

Injection Volume

25 μL

Elution Order

Ibuprofen, CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, Δ9 - THC, Δ8 -THC, CBL, CBC, THCA, CBCA, CBLA

Quantitating Cannabinoids in Edible Chocolates

Figure 1: A sample chromatogram (228 nm) of a 25 µg/mL calibration standard, with all target peaks labeled with identity and retention time.

Quantitating Cannabinoids in Edible Chocolates

Calculations a) Phytocannabinoid concentrations (% w/w) are calculated using the calibration parameters generated by the processing software with each sample batch analyzed. b) Sample and solvent masses are corrected as necessary. For example, the actual mass of sample or methanol used for extraction divided by the nominal amount – either 0.5 g of sample or 7.91 g for 10 mL methanol at 25 °C, respectively. c) Expected concentrations of cannabinoids and ibuprofen are outlined below (na – not applicable):

Sample type Phytocannabinoid (µg/mL) Ibuprofen (µg/mL) Unknown unknown 1 Method Blank na 1 ICS 5 5 RRS incurred + 1 2

G. Quality Control a) Calibration Check: A previous calibration function may be used provided a freshly prepared ICS meets accuracy specification of ±20% for all analytes. b) Calibration: If the calibration check does not meet specification, the detector response must be calibrated with a minimum of five calibration standards. The accuracy of each calibration point used for each analyte must be ±20% its expected value. c) Peak Detection: If a peak is identified by the data processing software, confirm its retention time by comparing samples to calibrators. d) Continuing Calibration Verification: Reinject one of the calibration standards at least once per sequence and then at a frequency of 5%. Recovery shall be between 80 - 120%. e) Method Blank: Analyze one per sample batch. Each analyte peak detected shall be below its corresponding Method Detection Limit (MDL). f) RRS: Analyze once per sample batch. A blank matrix is fortified at 1 μg/mL with all target analytes. Recovery shall be between 70 - 130%. Blank subtraction may be required depending on the incurred analytes in a given sample. g) RRS Duplicate: The RRS is always performed in duplicate. The relative percent difference (RPD; see below) of the RRS and RRS Duplicate must be < 30% for all target analytes. , % = 100 × | − |̅ , h) ICS: Analyze once per worksheet. Recovery must be between 70 and 130%. Must be prepared independently from calibration standards. i) Ibuprofen Check Standard Recovery: Recovery must be between 90 - 110% Ibuprofen (0.9 - 1.1 µg/mL in regular samples). H. Single-Laboratory Validation Study Design

Quantitating Cannabinoids in Edible Chocolates

a) Blank extracts were fortified with the 17 phytocannabinoid standards at 0.008% w/w (low), 1% w/w (mid), or 4% w/w (high) after the final dilution to facilitate method validation using commercially available analytical standards. There was no cost-effective way to complete this validation by fortifying chocolates prior to extraction. b) The absolute area response of all analytes in a blank sample analysed immediately after a calibrator were required to be ≤ 0.1% of the absolute area of the same analytes in the high calibrator when carry-over was evaluated. c) Chromatographic resolution (R s ) is calculated using representative calibration samples, with an Rs of at least 1.5 being desirable. = 1.18 × 2 − 1 2 + 1 Where t 2 (nearest eluting peak) and t 1 (target peak) are retention times and w 1 and w 2 are the peak widths at half-height of the analyte of interest and the nearest significant chromatographic peak, respectively. d) Recovery is determined by fortifying blank samples at low, mid and high concentrations, with five replicates at each level in milk, dark and white chocolate. e) Repeatability (RSD r ) is determined for each matrix at 0.008%, 1% and 4% w/w using five replicate extractions of fortified matrix f) Intermediate precision (two analysts, RSD R ) is calculated over three days with five replicate extracts at 0.008% and 1% w/w for each matrix. The intermediate precision at 4% w/w was not evaluated due to the associated costs. g) The method detection limit (MDL), defined as the minimum concentration of analyte that can be reported with 99% confidence that a measured concentration is distinguishable from a blank sample, is found by fortifying a total of seven replicates of each matrix across three days of validation at a concentration that yields a signal-to-noise of 3-5. Two analysts contribute to the preparation of extracts. The MDL for a given analyte is calculated with the following equation: , % / = × Where t is 3.134, the Student’s t -value for a single-tailed 99 th percentile t statistic and standard deviation estimate with 6 degrees of freedom, and S s is the standard deviation of the seven fortified replicate samples for each matrix. h) The method reporting limit (MRL), defined as the lowest concentration level reported for a given test method (which must be equal to or greater than the MDL), is calculated from seven replicates in each matrix prepared on a single day by a single analyst. The half range for the prediction interval of results (HR PIR ) is calculated from this data: = 3.963 × Where 3.963 is a constant for the seven extraction replicates and s is the standard deviation. i) This value is used to calculate the upper and lower prediction interval of results (PIR):

Quantitating Cannabinoids in Edible Chocolates

= ± × 100% j) If the upper PIR is less than 150% and the lower PIR is greater than 50%, the MRL is verified. I. Reference Materials Used a) No reference materials were available to support this work. b) Notwithstanding this limitation, the milk chocolate used was a rejected lot from a commercial manufacturer – it contains THC and CBD. Using procedures compliant with ISO/IEC 17034, this material was assessed for homogeneity and values were assigned for THC and CBD accordingly. These values were used as a reference point to demonstrate full method applicability for neutral phytocannabinoids.

Quantitating Cannabinoids in Edible Chocolates

Table 1: Method validation summary for phytocannabinoids in a dark chocolate.

Recovery (%)

0.008% w/w

1% w/w

4% w/w

Method Limits (% w/w)

ID Analyte

Calibration Range (% w/w)

Carry- Over (%)

R s

0.008% w/w

1% w/w

4% w/w

RSD r RSD R RSD r RSD R RSD r RSD R

MDL

MRL

1 2 3 4 5 6 7 8 9

CBC

0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1.4 3.8 2.3 2.3 3.1 3.1 2.3 2.3 1.4 4.6 3.3 1.5 1.5 3

105 93.5

101.4 102.9 3.04

6.21 5.32 5.21 7.99 6.15 7.14 6.06 7.84 3.88 6.53 6.53 7.19 3.57 5.47 4.77 7.53 7.1

0.73 1.27 0.96 1.13 1.05 1.79 1.06 0.74 0.63 0.88 1.63 1.46 1.05 1.73 0.94 1.4 1.5

3.68 5.38 3.39 4.28 2.45 3.59 3.03 5.32 1.61 1.97 4.99 3.61 1.99 2.51 1.63 1.97

0.57 0.58 0.54 0.66 0.44 0.44 0.68 0.82 0.86 0.46 0.44 0.64 0.54 0.57 0.51 1.32 0.54

na na na na na na na na na na na na na na na na na

0.00089 0.00051 0.00062 0.00159 0.00053 0.00115 0.00062 0.00134 0.00076 0.00063 0.00049 0.00129 0.00090 0.00066 0.00066 0.00080 0.00152

0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008

CBCA CBD CBDA CBDV

101.5 85.5

2.85

107.8 102.8

101.1 103.1 2.77

99.4 100.4 97.5

92.8 100 94.3

2.51 2.47 1.32 2.85 2.18 3.57 2.29

107 98.3

CBDVA

CBG

101.5 102.3 97.5 105.8 106.3 104.3 101.5

101.2 99.3 100.8 91.9 100.5 97.9 100.5 99.5

CBGA

CBL

10 11 12

CBLA CBN CBNA

100.3 103.3 2.37

2.63

13 d8-THC 14 d9-THC

100.8 100.7 3.84 101.4 100.2 2.28

102

15 16

THCA THCV

2 4

103.5

99.8 98.1 98.9

99.3

3.11 3.23

101 102

17 THCVA

3.3

92.1

2.8

4.2

Quantitating Cannabinoids in Edible Chocolates

Table 2: Method validation summary for phytocannabinoids in milk chocolate.

Recovery (%)

0.008% w/w

1% w/w

4% w/w

Method Limits (% w/w)

ID Analyte

Calibration Range (% w/w)

Carry- Over (%)

R s

0.008% w/w

1% w/w

4% w/w

RSD r RSD R RSD r RSD R RSD r RSD R

MDL

MRL

1 2 3 4 5 6 7 8 9

CBC

0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1.4 3.8 2.3 2.3 3.1 3.1 2.3 2.3 1.4 4.6 3.3 1.5 1.5 3

104.8 90.5 101.3 90.4 99.3 90.3 90.5 100.5 102.5 103.8 90.5 105.5 100.8 94.5 90.3 94 90

106.6 104.7 1.45

7.88 7.79 7.39 5.77 6.88 6.81 7.27 7.39 6.36 7.26 6.49 5.68 4.38 7.54 7.9

0.41 0.98 0.85 0.47 0.65 0.67 0.55 0.65 0.55 0.61 0.57 0.68 1.09 0.46 0.5 0.67 0.5

3.54 4.59 2.56 3.73 2.33 3.11 2.71 4.49 1.06 1.95 4.56 3.31 1.29 2.44 1.67 1.13 3.78

0.53 1.11 0.65 0.89 0.54 0.86 0.72 1.04 0.73 0.75 0.59 0.89 0.72 0.66 0.98

na na na na na na na na na na na na na na na na na

0.00234 0.00080 0.00090 0.00113 0.00054 0.00119 0.00108 0.00117 0.00097 0.00139 0.00060 0.00069 0.00097 0.00151 0.00096 ^ ^

0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008

CBCA CBD CBDA CBDV

91.6

89.4

2.86

105.8 104.5 0.71

91.2

96.2

0.84

101.5 101.8 1.91

CBDVA

91.4

96.9

1.52

CBG

101.8 101.2 1.85

CBGA

90.8 98.1

95.3 98.7

1.85 1.42

CBL

10 11 12

CBLA CBN CBNA

100.3 102.4 1.93 107.9 103.8 1.93

91.8 102.9 101.2

2.6

13 d8-THC 14 d9-THC

103 102

2.69 0.91

15 16

THCA THCV

2 4

100.9 102.4 1.41

100.2 100.3

6.8

0.7

17 THCVA

3.3

91.1

3.72

6.53

0.94

^ calculation of CBD and d9-THC MDL and MRL values were not possible due to presence of incurred analyte in this matrix. The MRL has been inferred based on performance in other matrices and on the performance of other neutral phytocannabinoids in this matrix.

Quantitating Cannabinoids in Edible Chocolates

Table 3: Method validation summary for phytocannabinoids in white chocolate.

Recovery (%)

0.008% w/w

1% w/w

4% w/w

Method Limits (% w/w)

ID Analyte

Calibration Range (% w/w)

Carry- Over (%)

R s

0.008% w/w

1% w/w

4% w/w

RSD r RSD R RSD r RSD R RSD r RSD R

MDL

MRL

1 2 3 4 5 6 7 8 9

CBC

0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5 0.004 - 5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1.4 3.8 2.3 2.3 3.1 3.1 2.3 2.3 1.4 4.6 3.3 1.5 1.5 3

103.3 91.5 108.8 102.3 102.5 97.3 99.3 99.8 97.3 104.5 107.5 104.8 104.3 105.5 101.8 102 99.5

107.6 104.3 4.06

6.96 4.36 6.99 5.14 5.64 7.03 4.67 6.84 7.62 5.66 7.84 5.06 6.94 7.89 7.75 5.3 4.3

0.6

3.94 4.53 3.48 3.16 2.99 3.14 3.03 3.99 2.78 2.42 5.63 3.16 2.54 2.36 2.7

0.09 0.97

na na na na na na na na na na na na na na na na na

0.00147 0.00065 0.00074 0.00101 0.00087 0.00074 0.00087 0.00077 0.00077 0.00060 0.00108 0.00087 0.00152 0.00118 0.00117 0.00133 0.00110

0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008

CBCA CBD CBDA CBDV

91.5

87.2

2.39

0.56 0.57 0.35 0.61 0.61 0.55 0.47 0.53 0.58 0.83 1.78 1.47 0.24 0.37 0.5 0.6

106.2 104.3 2.49

0.3 0.7

91.7

2.28

102.2 101.2 3.24

0.39 0.97 0.21 1.22 0.35 0.17 1.09 0.38 0.19 0.67 0.14 0.95 0.5

CBDVA

95.5

3.28

CBG

102.3 100.6 2.92

CBGA

91.3 98.8 101

93.6 98.3

1.18 3.53

CBL

10 11 12

CBLA CBN CBNA

100.8 1.12

108.9 104.5 2.66

2.27 2.28

13 d8-THC 14 d9-THC

102.9

102

100.9 101.2 1.34 101.4 100.9 3.71

15 16

THCA THCV

2 4

101.2 99.8

3.6

3.3

17 THCVA

3.3

91.4

93.9

1.29

3.37

AOAC Method Submission Form

Submission Date

2020-08-11 12:39:24

Submitter Name

Alana Greaves

Submitter Email

alana.greaves@canopygrowth.com

Organization

Canopy Growth Corporation

Method Type

Cannabis

Resubmission?

Method Name

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

Method Author(s)

Alana Greaves, Jessica Gauthier, Ben Geiling

Method Applicability

Applicable for determination of 0.005 – 6.25 % w/w cannabinoids in dark, milk, and white chocolate. For some cannabinoids, this range may be extended even further based on instrument linearity. This method accurately quantifies d9-THC, CBD, d9-THCA, CBDA, CBN, and d8-THC, and can precisely monitor an additional 9 cannabinoids. This method has been validated to include chocolate bars, chocolate chips, and chocolate truffles, but does not include chocolates with nuts, fruit, or other ingredients which will not fully homogenize upon melting. This method is suitable for HPLC-DAD as well as HPLC-HDR-DAD. Chocolate samples are melted and extracted with 90:10 methanol:chloroform. After a lipid-removal step, filtered samples are analyzed by gradient reverse- phase high-pressure liquid chromatography with a diode array detector.

A. Principle

B. Apparatus & Materials

(a) High-pressure liquid chromatograph (HPLC) – Liquid chromatograph with automated sampler, quaternary gradient pump, de-gas module, column heater, and diode array detector (DAD) or high dynamic range DAD (HDR-DAD). (Agilent Corporation, 5301 Stevens Creek Blvd., Santa Clara, CA, 95051. Tel: 1- 800-227-9770)

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.

Table 1. HPLC mobile phase gradient. HPLC Mobile Phase Pump A Deionized water Pump B Methanol Pump C Acetonitrile + 0.1% (v/v) phosphoric acid Pump D Deionized water + 0.1% (v/v) phosphoric acid Time (min) Pump A (%)1 Pump B (%) Pump C (%) Pump D (%)

0.0 0.0 0.0 66.0 34.0 7.5 0.0 0.0 66.0 34.0

10.5 0.0 15.0 66.0 19.0 14.0 0.0 15.0 66.0 19.0 15.0 0.0 34.0 66.0 0.0 21.0 0.0 34.0 66.0 0.0 22.0 0.0 0.0 66.0 34.0 25.0 0.0 0.0 66.0 34.0 1 Pump A is used for a seal wash.

ii. DAD Conditions – Flow cell 3 mm; DAD wavelength 215 nm; DAD slit width 4 nm. iii. HDR-DAD Conditions – Two flow cells connected in series for increased linear range: (1) 60 mm (2) 3.7 mm. DAD wavelength 215 nm; DAD slit width 4 nm. iv. 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 free of sample carry-over. (b) Column – Agilent Poroshell 120 EC-C18; 4.6 x 250mm x 2.7µm, with guard column of matching packing material; 4.6 x 5mm x 2.7µm. (c) Bead bath – Maintaining 65°C ± 2°C. (d) Ultrasonic water bath – With built-in timer for automatic shut-off. (e) Ultra low-temperature freezer – Maintaining -80°C ± 2°C. (f) Centrifuge – Maintaining a speed of 1000 x g.

C. Reagents

(a) Extraction Solution. – 90:10 methanol:chloroform solution. Add 100 mL of HPLC-grade chloroform to a 1L volumetric flask. Fill to the mark with HPLC- grade methanol. (b) Mobile Phase A. – Deionized water. (c) Mobile Phase B. – HPLC-grade methanol. (d) Mobile Phase C. - Acetonitrile + 0.1% (v/v) phosphoric acid. 1.175 mL of phosphoric acid (85% v/v), or equivalent is added to a 1L volumetric flask using a verified pipette. Fill to the mark with HPLC-grade acetonitrile. (e) Mobile Phase D. – Deionized water + 0.1% (v/v) phosphoric acid. 1.175 mL of phosphoric acid (85% v/v), or equivalent is added to a 1L volumetric flask using a verified pipette. Fill to the mark with deionized water. (f) Analytical Standards. – Certified Reference Materials; 1 mg/mL; Cerilliant Corporation. i. d9-THC. Δ9- tetrahydrocannabinol ii. CBD. Cannabidiol iii. d9-THCA. Δ9-tetrahydrocannabinolic acid iv. CBDA. Cannabidiolic acid v. CBN. Cannabinol x. CBG. Cannabigerol xi. CBE. Cannabielsoin xii. THCVA. Tetrahydrocannabivarinic acid xiii. CBL. Cannabicyclol xiv. CBC. Cannabichromene xv. CBCA. Cannabichromenic Acid (g) Analytical Standards. – Certified Reference Materials; 2.5 mg; Toronto (a) Preparation of calibration curve. – Prepare a calibration curve in methanol from certified analytical standards for each cannabinoid of interest. Calibration curves may be prepared individually, or mixed. The linear range of each compound’s prepared calibration curve should range from 0.1% w/v to 0.00001% w/v. Points at either extremity of the curve which reduce the curve’s linearity (R2 < 0.99), or whose signal-to-noise ratio is less than 10 should not be used. All calibration curve solutions should be prepared using a gas-tight syringe using methanol as a diluent. Calibration curves are plotted as Peak Area vs. Concentration (% w/v), with the x-intercept set to 0.0 and the line of best fit following the equation Peak area = (Slope)(Concentration). The instrument’s minimum linear range must be equal to 0.6 – 400 ppm to accurately capture cannabinoid concentrations of 0.008 – 5% w/w in chocolate samples. (b) Preparation of test samples. i. For solid chocolate, including chocolate bars and chocolate chips – Homogenize the solid chocolate sample using a mortar and pestle. Accurately weigh 0.08000 g of chocolate (with a tolerance of +5%) into a glass test tube and record the mass. ii. For chocolate truffles – Place the entire truffle into a closed container, such as a 50 mL centrifuge tube. Place in a 65°C bead bath until the chocolate is melted, about 10 minutes. Using a lab spatula or stir bar, thoroughly mix the melted chocolate. Accurately weigh 0.08000 g of chocolate (with a tolerance of +5%) into a glass test tube and record the mass. Note: This method has been validated for chocolate truffles which can fully homogenize upon melting, such as those dusted with cocoa powder or coated with solid chocolate. This method does not apply to chocolate truffles with nuts, fruit, or other ingredients which will not fully homogenize when melted. Research Chemicals i. CBE. Cannabielsoin vi. d8-THC. Δ8-tetrahydrocannabinol vii. CBDVA. Cannabidivarinic Acid viii. CBDV. Cannabidivarin ix. CBGA. Cannabigerolic acid

D. Preparation of Test Samples / Standard Solutions

E. Determinations

(a) Preparation of test sample for HPLC analysis. – Heat the weighed sample using a 65°C bead bath until the sample has fully melted, about 5-7 minutes. Before the sample cools, immediately add 10 mL of the Extraction Solution to the test tube. Vortex the sample for 30 seconds. Sonicate the 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. 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.

H. Calculations

"Conc.in test sample (% w/w) = " "Peak Area in test sample" /"Slope" " × " "10mL" /"Test sample mass (g)"

Reference Materials Used:

(a) All calibration curves are prepared using certified reference materials at a concentration of 1 mg/mL (Cerilliant Corp.) or else purchased powdered (Toronto Research Chemicals). AOAC Proposed Method - Cannabinoid potency in chocolate.docx AOAC Single Laboratory Validation - Cannabinoid potency in chocolate.docx Copyright Transfer Form - Signed.pdf

Upload manuscript and other supporting information here. Multiple files allowed.

Author Notes / Comments

Attached documents: (1) Proposed method (2) Single Laboratory Validation manuscript (3) Copyright transfer form

AOAC Proposed Method SMPR 2017.019

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

2 3

Chromatography

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)

21 22 23 24

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.

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

34.0 34.0

1 Pump A is used for a seal wash.

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