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

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AOAC Analytical Methods Week July 25 – 29, 2022 ERP for Chemical Contaminants in Cannabis & Hemp (CASP)

AOAC INTERNATIONAL 2275 Research Blvd, Suite 300 Rockville, MD 20850

Method Review REVIEW FORMS DUE: FRIDAY, JULY 22, 2022

Table of Contents

PRELIMINARY ITEMS AOAC Policies and Procedures AOAC SMPR 2021.010 AOAC ERP FOR CHEMICAL CONTAMINANTS IN CANNABIS METHHODS AGENDA EXPERT REVIEW PANEL FIRST ACTION METHOD REVIEWS REVIEW OF CANDIDATE METHOD FOR FIRST ACTION STATUS Protocol – Quantitation of Mycotoxins in Cannabis Biomass and Cannabis Derived Products AOAC 2022.XX - Quantitation of Mycotoxins in Cannabis Biomass and Cannabis Derived Products

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ACCESS TO REVIEW FORM FOR CANDIDATE METHODS https://form.jotform.com/210776414508153

ERP Premeeting Method Review Book

ACCESS TO AOAC BYLAWS, POLICIES & PROCEDURES, AND STRATEGIC PLAN AOAC INTERNATIONAL Bylaws (201 9 ) The excerpt from the Bylaws below is Article VIII which pertains to the Official Methods of Analysis and the Official Methods Board. ARTICLE VIII Official Methods of Analysis The Board of Directors (BoD) is empowered to develop written policies and procedures for the study, adoption, and change in status of the Official Methods of Analysis of AOAC INTERNATIONAL. Implementation of the policies and procedures shall be delegated to an Official Methods Board (OMB). Section 1. Composition of the Official Methods Board The Official Methods Board shall consist of a chair and a vice chair, and members who are recommended by the chair. The chair, vice chair and members are appointed by the President of AOAC INTERNATIONAL. The OMB shall be composed of members representing a balance of government, industry, and academia as appropriate to the scope of the group and shall not be dominated by any single interest. Section 2. Purpose of the Official Methods Board The OMB shall serve the Association in a scientific and advisory capacity on methods and the process of their adoption. The OMB shall be responsible for implementation of procedures adopted by the BoD, according to the principles in section 3 below. Section 3. Principles of the Official Methods Program A. Adequate records of technical data, discussions, and decisions on the study, adoption, and change of status of Official Methods of Analysis shall be maintained for a reasonable time. B. Timely notice of proposed method studies, adoption, or change in status shall be published in an Association publication that is circulated to the members. C. Opportunity shall be provided for materially interested parties to submit input during method study and adoption procedures and to submit comments on the adoption, use of, or change in status of specific methods. D. Methods submitted to the OMB for inclusion in the OMA shall be thoroughly studied, scientifically reviewed, and available in published form prior to adoption as Final Action by the OMB. E. The OMB shall adopt methods as Final Action. AOAC INTERNATIONAL Policy on Antitrust Below is an excerpt from the Policy that are the guidelines to be followed. Since the individual has an important responsibility in ensuring antitrust compliance in AOAC activities, everyone should read and heed the following guidelines. 1. Don't make any effort to bring about or prevent the standardization of any method or product for the purpose or intent of preventing the manufacture or sale of any method or product not conforming to a specified standard. 2. Don't discuss with competitors your own or the competitors' prices, or anything that might affect prices such as costs, discounts, terms of sale, distribution, volume of production, profit margins, territories, or customers. 3. Don't make announcements or statements at AOAC functions, outside leased exhibit space, about your own prices or those of competitors. 4. Don't disclose to others at meetings or otherwise any competitively sensitive information.

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ERP Premeeting Method Review Book

5. Don't attempt to use the Association to restrict the economic activities of any firm or any individual. 6. Don't stay at a meeting where any such price or anti‐competitive talk occurs. 7. Do conduct all AOAC business meetings in accordance with AOAC rules. These rules require that an AOAC staff member be present or available, the meeting be conducted by a knowledgeable chair, the agenda be followed, and minutes be kept. 8. Do confer with counsel before raising any topic or making any statement with competitive ramifications. 9. Do send copies of meeting minutes and all AOAC related correspondence to the staff member involved in the activity. 10. Do alert the AOAC staff to any inaccuracies in proposed or existing methods and statements issued, or to be issued, by AOAC and to any conduct not in conformance with these guidelines. AOAC INTERNATIONAL Policy on Use of Association Name, Initials, Identifying Insignia, Letterhead and Business Cards Below is an excerpt from the Policy statement. The Executive Director, in accordance with the above stated policy, is authorized to process, approve, fix rules, and make available materials containing the Association name and insignia. It should be noted that neither the Association's name nor its insignia nor part of its insignia may be incorporated into any personal, company, organization, or any other stationery other than that of the Association; nor may any statement be included in the printed portion of such stationery which states or implies that an individual, company, or other organization is a Member of the Association 1. Do avoid the appearance as well as the fact of a conflict of interest. 2. Do make written disclosure of any material interest which may constitute a conflict of interest or the appearance of a conflict of interest. 3. Do not accept payment or gifts for services rendered as a volunteer of the Association without disclosing such payment or gifts. 4. Do not vote on any issue before an AOAC decision making body where you have the appearance of or an actual conflict of interest regarding the recommendation or decision before that body. 5. Do not participate in an AOAC decision making body without written disclosure of actual or potential conflicts of interest in the issues before that body. 6. Do not accept a position of responsibility as an AOAC volunteer, without disclosure, where the discharge of the accepted responsibility will be or may appear to be influenced by proprietary or other conflicting interests. AOAC INTERNATIONAL Policy on Volunteer Conflict of Interest Below is an excerpt from the Policy of Do’s and Don’t’s

July 2022

AOAC INTERNATIONAL Strategic Plan ERP Premeeting Method Review Book

Vision

Global confidence in consensus based analytical solutions for food safety, food integrity, and public health.

Mission

AOAC INTERNATIONAL ensures the safety and integrity of foods and other products that impact public health by convening government, industry and academia to develop and validate standards, methods and technologies.

Goals

Provide analytical solutions to current and emerging issues through standards development and trusted measurements. Attract and retain members and stakeholders through education, mentoring, networking and collaboration to grow and strengthen the association. Build and cultivate relationships to identify strategic opportunities for collaboration. Develop new and improve existing processes, products and services. Identify, strengthen, and grow revenue streams, while continuously optimizing resources, to ensure the association’s long-term success. Advance an effective leadership culture that promotes accountability among members and staff.

Analytical Excellence

Engagement

Partnerships

Core Programs

Sustainability

Governance

July 2022

December 10, 2019

ERP Premeeting Method Review Book

OFFICIAL METHODS OF ANALYSIS OF AOAC INTERNATIONAL FIRST ACTION TO FINAL ACTION GUIDANCE FOR METHOD AUTHORS

ethods published in the Official Methods of Analysis of AOAC INTERNATIONAL (OMA) undergo scientific validation and evaluation. Methods in good standing are designated as First Action status upon initial adoption by an AOAC expert review panel (ERP). Methods are designated as Final Action status upon

consensus of the AOAC Official Methods Board.

AOAC First Action status methods are tracked for a maximum of two (2) years and then evaluated for a status change. ERPs track the methods during the two year period during which the method is expected to be used and during which time, method reproducibility can be assess (if not completed), method performance feedback from method users, laboratory proficiency testing results, method modifications, etc… ERPs may make suggestions following the initial adoption of a method to be completed during the two ‐ year tracking period. At the end of the two ‐ year period, the ERP reviews the method and all information gathered during the two ‐ year period to verify that a final version of the method is substantiated. The guidance below is for authors of the published manuscripts of the methods published in the OMA that are under consideration for status changes.

Method authors will need to consider the following: 1. Review the method as it is published in the OMA a. If there are changes, request an MS Word tracking version of the method from AOAC by contacting Deborah McKenzie at dmckenzie@aoac.org. 2. Review the ERP report from when the method underwent its last review. a. Are there any ERP requirements that were agreed upon for the method? These may include additional data submission, etc…. 3. Collect performance feedback on the method. a. Can come from collaborators and/or other method users, PT data may also be included. 4. Has method reproducibility been assessed and reviewed by the ERP? a. If this was completed as part of the initial adoption of the method. b. If this has been recently collected, then you need to do the following: i. Draft a manuscript in AOAC format. ii. It is highly recommended that the manuscript and raw data

files will require a review by AOAC Committee on Statistics

METHOD AUTHOR SUBMISSION PACKAGES 1. Submission package for Final Action status consideration must include the following individual files: a. AOAC formatted manuscript with reproducibility b. Separate files for tables and figures c. Copy of the published method with tracked changes for the final version of the method d. Presentation to the ERP 2. Submission package for Continuance of First Action status must include the following: a. Written rationale for continuance with a timeframe b. Any supporting information such as proposal for additional or continuing studies. 3. Submission package for Repealing a method from the OMA a. Written rationale for removing the method b. Any supporting information

Version: 11 ‐ 2020

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ERP Premeeting Method Review Book

Method developers may choose one or more of the suggested matrices. Method developers must specify the matrix or matrices used. Limit of detection (LOD) .—Smallest measured concentration of an analyte from which it is possible to deduce the presence of the analyte in the test sample with acceptable certainty. There are several scientifically valid ways to determine LOD, and any of these could be used as long as a scientific justification is provided for their use. For examples, see Guidance for Industry Studies to Evaluate the Metabolism and Residues Kinetics of Veterinary Drugs in Food-Producing Animals: Validation of Analytical Methods Used in Residue Depletion Studies , U.S. Food and Drug Administration (2015). Limit of quantitation (LOQ) .—Minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result [refer to Appendix F: Guidelines for Standard Method Performance Requirements (2019) 21st Ed., Official Methods of Analysis of AOAC INTERNATIONAL , http://www.eoma.aoac.org/ app_f.pdf]. Accordingly, LOQ is the lowest concentration or mass that can be reported as a numerical value. Scientific justification for the procedure used to determine LOQ should be provided. Measurement uncertainty. — Non-negative parameter characterizing the dispersion of the values being attributed to the measured value [ Guidelines for Validation of Chemical Methods for FDA Foods Program (2019) 3rd Ed., U.S. Food and Drug Administration]. Quantitative .—Method of analysis for which response is the amount of the analyte measured either directly (enumeration in mass or volume) or indirectly (color, absorbance, impedance, etc.) in a certain amount of sample. Recovery .—Fraction or percentage of fortified or incurred 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 ). Reproducibility .—Standard deviation or relative standard deviation calculated from among-laboratory data; expressed as the reproducibility standard deviation (SD R ) or % reproducibility 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) Refer to: Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements (2019) 21st Ed., Official Methods of Analysis of AOAC INTERNATIONAL , Rockville, MD, USA, http://www. eoma.aoac.org/app_f.pdf ISO 17034:2016 General requirements for the competence of reference material producers (2016) International Organization for Standardization, https://www.iso.org/obp/ui/#iso:std:iso:17034:en relative standard deviation (% RSD R ). 5 Method Performance Requirements See Tables 1 and 2.

AOAC SMPR ® 2021.010

Standard Method Performance Requirements (SMPRs ® ) for Quantitative Analysis of Mycotoxins in CannabisBiomass and Cannabis-Derived Products Intended Use: Testing of Cannabis Biomass and Cannabis-Derived Products 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 stakeholders 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 methods 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 [refer to Appendix F: Guidelines for Standard Method Performance Requirements (2019) 21st Ed., Official Methods of Analysis of AOAC INTERNATIONAL , http://www. eoma.aoac.org/app_f.pdf]. 2 Applicability Method, or a suite of methods, to identify and quantify ochratoxin A, aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 , and aflatoxin G 2 in cannabis biomass, hemp, and/or cannabis-derived products. Ochratoxin A, aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 , and aflatoxin G 2 are required analytes. Testing of other toxins in addition to the required toxins can be submitted. 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 Analytes: Total aflatoxins .—Sum of aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 and aflatoxin G 2 . Testing other toxins in addition to this list may be possible. Matrices: Cannabis biomass .—Plant material from Cannabis spp. and its chemical varieties or chemotypes, for example, flower, trim, and fiber. Hemp is included in this definition of cannabis biomass. Cannabis-derived products.— Products or extracts derived from cannabis plant material. Derivative products include but are not limited to ingestible/edibles, inhalation products, concentrates and extracts, and hempseed and hempseed oil. Ochratoxin A.— CAS 303-47-9. Aflatoxin B 1 .— CAS 1162-65-8. Aflatoxin B 2 .— CAS 7220-81-7. Aflatoxin G 1 .— CAS 1165-39-5. Aflatoxin G 2 .— CAS 7241-98-7.

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ERP Premeeting Method Review Book

ISO Guide 80:2014 Guidance for in-house preparation of quality control materials (QCMs) (2014) International Organization for Standardization, https://www.iso.org/obp/ ui/#iso:std:iso:guide:80:ed-1:v1:en 8 Validation Guidance All claimed matrices shall be evaluated. Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis (2019) 21st Ed., Official Methods of Analysis of AOAC INTERNATIONAL , Rockville, MD, USA, http://www.eoma.aoac.org/app_d.pdf Appendix K: Guidelines for Dietary Supplements and Botanicals (2019) 21st Ed., Official Methods of Analysis of AOAC INTERNATIONAL , Rockville, MD, USA, http://www.eoma.aoac. org/app_k.pdf Guidelines for Validation of Chemical Methods for FDA Foods Program (2019) 3rd Ed., U.S. Food and Drug Administration, https://www.fda.gov/media/81810/download Guidance for Industry Studies to Evaluate the Metabolism and Residues Kinetics of Veterinary Drugs in Food-Producing Animals: Validation of Analytical Methods Used in Residue Depletion Table 1. Target levels for analytes to be included in method; example regulatory limits canbe found in Appendix 1 Compound Target level, µg/kg a Target LOQ, µg/kg Ochratoxin A 20 Less than target level Aflatoxin B 1 5 Less than target level Total aflatoxins b 20 Less than target level a Methods with lower target levels will be accepted. LOQ should be less than the target level a method developer is using for their method. b Total aflatoxins = Sum of aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 , and aflatoxin G 2 .

Table 2. Specific method performance requirements Parameter Requirement LOQ, µg/kg

Less than target level in Table 1

Applicable range, µg/kg

Must be stated

LOD, µg/kg

Must be determined and method of determination detailed For example: 3.3 × standard deviation of blank sample (refer to ICH Q2 guidance a )

Recovery, %

60–120

Repeatability (RSD r ), %

≤22 for 1 to <100 µg/kg ≤11 for 100–999 µg/kg ≤44 for 1–999 µg/kg

Reproducibility (RSD R ), % Measurement uncertainty

Must be determined and method of determination detailed

a ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology (1995) International Council for Harmonization, https://www. ema.europa.eu/en/documents/scientific-guideline/ich-q-2-r1-validation- analytical-procedures-text- methodology-step-5_en.pdf

Studies (2015) U.S. Food and Drug Administration, https://www. fda.gov/media/78356/download ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology (1995) International Council for Harmonization, https://www.ema.europa.eu/en/documents/scientific-guideline/ich- q-2-r1-validation-analytical-procedures-text-methodology-step-5_ en.pdf Developed by AOAC Cannabis Analytical Science Program (CASP) Product-Centric Working Group (joint working group on Cannabinoids and Consumables and Chemical Contaminants). Approved by AOAC CASP stakeholders on July 9, 2021. Final version date: July 9, 2021. Effective date: July 9, 2021.

Appendix 1. Example regulatory limits based on cannabis, hemp, or noncannabiscommodities Example regulatory limits, a µg/kg

Total aflatoxins + ochratoxin A

Ochratoxin A

Aflatoxin B 1

Aflatoxin B 2

Aflatoxin G 1

Aflatoxin G 2

Total aflatoxins b

Canada

<20

4 4

Australia

California, USA Colorado, USA Oregon, USA

≤20

≤2 <5

≤20 <20 <20 ≤20

<20

Pennsylvania, USA

≤5

≤20

Florida, USA Illinois, USA

≤20 ≤20

a Based on cannabis, hemp, or noncannabis commodities at the time of publication (October 2021). b Total aflatoxins = Sum of aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 , and aflatoxin G 2 .

July 2022

AOAC ERP FOR CHEMICAL CONTAMINANTS IN CANNABIS METHODS AOAC ANALYTICAL METHODS WEEK JULY 25 - 29, 2022 DRAFT ERP MEETING AGENDA

Wednesday, July 27, 2022 12:30PM ET – 2:30PM ET

Chair: Andrew Pham (Epic Solutions and Analytics – Environmental Services)

WELCOME AND INTRODUCTIONS (Pham) Andrew Pham will welcome attendees and AOAC will establish the presence of an ERP quorum via roll call by Allison Baker (AOAC).

II.

ERP MEETING PROCEDURES OVERVIEW (McKenzie/Baker) McKenzie will provide an overview of procedures for the ERP meeting and method review.

III. REVIEW OF CANDIDATE METHOD FOR FIRST ACTION STATUS* (Pham) For the ERP’s consideration, a validation report for the Quantitative Analysis of Mycotoxins in Cannabis Biomass and Cannabis Derived Products submitted by R-Biopharm Rhone in conjunction with Canopy Growth Corporation for First Action consideration. The assigned ERP reviewers, Dr. Tetsu Goto, Dr. Salvatore Parisi, Dr. Brent Wilson, and Dr. Julie Kowalski will share their reviews followed by ERP deliberation using AOAC SMPR 2021.010 . The ERP will render a decision on the method regarding a First Action adoption. IV. ADJOURNMENT (Pham)

*Items requiring a vote ERP for Low Lactose Methods Meeting Agenda – version 1

Friday, July 22, 2022 AOAC CASP Expert Review Panel: First Action Method Review

Reviewer Information Name

JULIE KOWALSKI

E-mail

julie@kowalskiscience.com

Organization

JA Kowalski Science Support LLC

Tip: Not able to complete this review on one sitting? Scroll to the bottom and click "save" at any time. Questions? Contact Allison Baker, Standards Coordinator, CASP at abaker@aoac.org. Method Review Title of Method Quantitation of Mycotoxins in Cannabis Biomass and Cannabis Derived Products AOAC Candidate Method Number (e.g. ALN-01) AOAC 2022.XX Applicable SMPR AOAC SMPR 2021.010 Preliminary Questions: Manuscript Format and Completeness

Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results? Are the figures and tables sufficiently explanatory without the need to refer to the text? Are all the tables and figures pertinent? Could some tables and figures be omitted and covered by a simple statement? Are the references complete and correctly annotated?

Yes

Does the method contain adequate safety precaution references and/or statements? I. Summary of the Method I. Summary of the Method

Yes

This method is for the quantitative determination of aflatoxins (B1, B2, G1, G2) and Ochratoxin A using immunoaffinity SPE style cleanup tubes and analysis using post column derivatization and HPLC- Fluorescence detection. Immunoaffinity cleanup selectively retains the mycotoxins, coextractives are eluted, then analytes (mycotoxins) are eluted. Then, the sample is prepared for injection into the HPLC/FL instrument. Post column derivatization is used to derivatize mycotoxins to increase signal ultimately allowing for better (lower) detectability. A large variety of cannabis matrices were tested. II. Review of the Method Only: II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. Yes. All required analytes and a sufficient variety of matrices were evaluated including flower, vape liquid, distillate, edible oils (MCT), chocolate, gummies, beverage. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. Not is all aspects: 1) Applicable range was was note clearly defined or stated for any analytes. 2) LOD determination method of determination was not detailed. 3) Measurement uncertainty was not detemined and method of determination was not detailed. One sentance was provided with, I think, the formula they intended to use for standard uncertainty calculation. I did not see how this related to data and no values provided. 4) System sutiability tests and/or analytical quailty control was not discussed beyond the checking of matrices before use in validation studies. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. yes 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). yes III. Review of Information in Support of the Method III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. No: Based on supporting information that was used the same method as the Method submitted, I do not know how LOD, LOQ, measurement uncertainty are determined so I can not determine if SMPR definitions of these terms were applied appropriately. 2. Is there information demonstrating that the method meets the SMPR Method Performance 2

Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. Yes (CRMs used) 3. Is there information demonstrating that the method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. Not is all aspects: 1) Applicable range was was note clearly defined or stated for any analytes. 2) LOD determination method of determination was not detailed. 3) Measurement uncertainty was not detemined and method of determination was not detailed. One sentance was provided with, I think, the formula they intended to use for standard uncertainty calculation. I did not see how this related to data and no values provided. 4) I am unclear about recovery data for aflatoxins B2, G1 and G2 for "betwee-day" recovery experiments so i can not determine if they meet the SMPR requirement 60-120%. The method determines the concentration of each analyte separately but it appears recovery was summed for the 4 aflatoxins so recoveries for B2, G1 and G2 were not provided. 5) summaries of data were provided but raw data like concentration values or recoveries for each replicate was not provided 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? No 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. No. I did not see any indication of sytem suitability tests or controls were used other than checking matrices for incurred aflatoxins. needed: 1) reagent blank(s) 2) process blank 3) spike recovery check per sample preparation batch - at lowest level and, ideally at a mid concentration point 4) calibration curve is serial dilution which does not allow you to find errors in initial standard preparation (error is carried through curve). A check standard(s) either from independent preparation ideally from a second lot or second source. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. no 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. Not is all aspects: 1) in section A. Principle: says toxins are eluted with 100% methanol but later is written elution occurs with methanol then water. 2) ppb is used. This is ambiguous and should be changed to per g or per mL units 3 IV. General Submission Package IV. General Submission Package

3) abbreviations of analytes not defined well 4) sum of aflatoxins is not defined or calculation discussed 5) methanol drying step potentially use a bit more detail 6) confusing when sections of other unnamed documents were referenced 7) I could not find specifics on the IA cleanup products so could not correlate the method with supporting information 5. Based on the supporting information, what are the pros/strengths of the method? Pros: 1) cleanup works well to reduce coextractives 2) separation is acceptable for all analytes 3) highly sensitive 4) method sufficient for a wide variety of cannabis matrices with only small modifications for gummies and hydrophic matices 6. Based on the supporting information, what are the cons/weaknesses of the method? 1) serial dilution calibration curve with out QC measures to verify 2) lack of system suitability and QC samples 3) lack of data on methanol drying step. Was this vetted to determine if methanol was evaporated especially from "oily" matices? Was it possible to use higher concentrated CRMs to deliver spiking volumes at lower volumes so that the dilution from spiking was relatively insignificant? 4) Data on each replicate was not provided either in concentration or recovery. This eliminates the ability for us to determine if there are any trends, for example, carry over. Similarly, raw data like calibration curve information (model, weighting) was not provided. 5) somewhat labor intensive 6) immunoaffinity cleanup (SPE) is a more skilled lab technique 7) uses instrumentation very unlikely to be already part of a typical cannabis lab suite of instruments 8) Although meets SMPR, the calibration range for alfatoxins maybe lower than needed for some method users 7. Any general comments about the method? The method appears to work sufficiently for a wide range of products which is great. The use of instrumentation that is not at all common in most cannabis labs may prevent this from being adopted. The cannabis testing industry (in the US) is very economically challenging. Many labs are using LC- MS/MS (although with challenges) for aflatoxins and ochratoxin A. This method has great sensitivity largely due to post column derivatization. I guestion if this added sensitivity is needed for most cannabis testing based on regulatory limits. I think the sample preparation (IA cleanup) is very valuable because analysis without it, even on LC-MS/MS, is very challening. Recommendation for the Method V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale. I do not recommend this method as written primarily due to:

1) some lack of clarity (see comments to question 4) including little on data analysis 2) lack of QC samples 3) lack of access to raw data

Tuesday, July 19, 2022 AOAC CASP Expert Review Panel: First Action Method Review

Reviewer Information Name

Salvatore Parisi

E-mail

drparisi@inwind.it

Organization

Lourdes Matha Institute HMCT

Tip: Not able to complete this review on one sitting? Scroll to the bottom and click "save" at any time. Questions? Contact Allison Baker, Standards Coordinator, CASP at abaker@aoac.org. Method Review Title of Method Quantitation of Mycotoxins in Cannabis Biomass and Cannabis Derived Products AOAC Candidate Method Number (e.g. ALN-01) AOAC 2022.XX Applicable SMPR • AOAC SMPR® 2021.010 - Standard Method Performance Requirements (SMPRs®) for Quantitative Analysis of Mycotoxins in Cannabis Biomass and Cannabis-Derived Products Preliminary Questions: Manuscript Format and Completeness

Yes

Are the references complete and correctly annotated? Does the method contain adequate safety precaution references and/or statements?

Yes

I. Summary of the Method I. Summary of the Method From the SLV document: The test employs the use of an immunoaffinity column, containing a mixture of different monoclonal antibodies which are specific for aflatoxins and ochratoxin A. 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 incannabis derived products, however the method could be extended in the future should legislation for other mycotoxins be introduced. The use of a multi-mycotoxin IAC ensured good recovery and precision without matrix effects and the ability to apply a single method to a diverse range of matrixes. The method has been shown to have good specificity with sensitivity, recovery, and precision meeting the requirements by the EU for methods of analysis for aflatoxins and OTA in foodstuffs. The method is suitable for routine testing of cannabis and cannabis products to ensure they are free from or below the limits of aflatoxin and OTA contamination. II. Review of the Method Only: II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. Yes, the applicability of the method supports the applicability of the AOAC SMPR® 2021.010 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. Yes, the analytical technique(s) used in the method meet the AOAC SMPR® 2021.010. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. Yes, definitions specified in the AOAC SMPR® 2021.010 are used and applied appropriately in the method, although the ““Protocol for Quantitative Analysis of Mycotoxins in Cannabis Biomass and Cannabis Derived Products” needs to be adequately AOAC- formatted and re-written. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). No. Actually, I have not seen safety reviews or MSDS documents concerning used materials such as methanol, nitric acid, potassium bromide, potassium chloride... The presence of similar documents would help, as in previous ERP evaluations when speaking of different methods, also in the CASP ambit. On these bases, the sentences “the responsibility of the user of this protocol to establish appropriate safety and health practices” and “protective clothing, gloves, and safety glasses should be worn, and all standard and sample preparation stages should be carried out in a fume cupboard” are justified.

III. Review of Information in Support of the Method III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. Yes. I am speaking of the SLV study as published in "Food Additives and Contaminants". In particular, inter- and intra-day operative conditions have been described in detail, as expected. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. The “protocol” document states that there are not available reference materials. On the other hand, the use of spiking protocol allows the method to meet the SMPR Method Performance Requirements by means of in-house reference materials. 3. Is there information demonstrating that the method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. With reference to the already published SLV document, all supporting data (recovery, LOQ, LOD, RSDr) are shown in Tables 5, 6, and 7. In particular, please note the remarkable performance with relation to LOQ and LOD when speaking of ochratoxin, aflatoxin B1, and total aflatoxins. IV. General Submission Package IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? No. 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. Yes. According to the SMPR (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. Appropriate tests have been carried out as demonstrated in Tables 3 to 6. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. Yes. Suitable methods have to include blank check samples and check standards at the lowest point and midrange point of the analytical range. Appropriate tests have been carried out as demonstrated in the “Results and Discussion” section of SLV, and in Tables 3 to 6. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. The method (in the SLV document) is written clearly and concisely. However, it needs to be formatted according to AOAC Guidelines. 5. Based on the supporting information, what are the pros/strengths of the method? Generally speaking, good specificity with sensitivity, recovery, and precision. Excellent performance when speaking of LOQ and LoD results. It is a good and simple routine method for screening of cannabis and cannabis products.

6. Based on the supporting information, what are the cons/weaknesses of the method? The method considers the minimum number of mycotoxins according to the SMPR. This point can be a discussion and enhancement factor, in future (and the SLV document clearlt states it). In addition, all mycotoxins are clearly defined; however, CAS numbers could be reported for each analyte. 7. Any general comments about the method? The method is concise and clear, well written. However, it has to be AOAC-formatted. Recommendation for the Method V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale. I would recommend the adoption as a First Action method with publication in the Official Methods of Analysis of AOAC INTERNATIONAL provided that: - The manuscript is re-written in the AOAC format for publication in all parts, including references; - Some additional safety (material safety data sheets or similar) documents are mentioned (when speaking of reagents) - Additional information for mycotoxins (CAS numbers) are mentioned.

Saturday, July 16, 2022 AOAC CASP Expert Review Panel: First Action Method Review

Reviewer Information Name

Tetsuhisa Goto

E-mail

tetsuhisagoto@yahoo.co.jp

Organization

CSCJP

Tip: Not able to complete this review on one sitting? Scroll to the bottom and click "save" at any time. Questions? Contact Allison Baker, Standards Coordinator, CASP at abaker@aoac.org. Method Review Title of Method Analysis of aflatoxins and ochratoxin A in Cannabis and Cannabis Products by LC-Fluirescence detection/// AOAC Candidate Method Number (e.g. ALN-01) ML0982 Applicable SMPR AOAC SMPR 2021.010 Preliminary Questions: Manuscript Format and Completeness

Yes

Does the method contain adequate safety precaution references and/or statements?

I. Summary of the Method I. Summary of the Method Method itself may be fit for this analysis but supporting data is not satisfactory.

Need re-design study and take sufficient data. II. Review of the Method Only: II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. yes 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. yes 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. yes 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). Method does not written folowing OMA format III. Review of Information in Support of the Method III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. yes 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. No RMs was used. 3. Is there information demonstrating that the method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. No IV. General Submission Package IV. General Submission Package

1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? Need SLV using adequately prepared samples. Please show data using naturally contaminated samples. At the case of spike and recovery test, spiked sample should be kept at least over-night to allow the evaporation of solvent and maycotoxins well attach to the sample matrix. 200 uL is to much to spike, volume of spike solution need to reduce ideally less than 5% of the amount of matrix. If spiking need to be done using large amount like 200 uL for 1 g of matrix, amount of remaining solvent need to be checked. 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. See above 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. No. please see above 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. No 5. Based on the supporting information, what are the pros/strengths of the method? IAC clean up is generally well accepted in mycotoxin analysis field 6. Based on the supporting information, what are the cons/weaknesses of the method? Sample used in this study. 7. Any general comments about the method? Need additional study using appropriately prepared samples. Recommendation for the Method V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale. Some additional data is required because of the study design of the manuscript has problem. 1. Because natural contamination of aflatoxins and ochratoxin A are very heterogeneous therefore amount of sample (1.0 g) used in this study is too small especially flour sample.

Friday, July 22, 2022 AOAC CASP Expert Review Panel: First Action Method Review

Reviewer Information Name

Walter Wilson

E-mail

walter.wilson@nist.gov

Organization

NIST

Tip: Not able to complete this review on one sitting? Scroll to the bottom and click "save" at any time. Questions? Contact Allison Baker, Standards Coordinator, CASP at abaker@aoac.org. Method Review Title of Method Quantitative Analysis of Mycotoxins in Cannabis Biomass and Cannabis Derived Products AOAC Candidate Method Number (e.g. ALN-01) AOAC 2022.XX Applicable SMPR 2021.010 Preliminary Questions: Manuscript Format and Completeness

Yes

Does the method contain adequate safety precaution references and/or statements? I. Summary of the Method I. Summary of the Method

Yes

The submitted analytical method focuses on the quantitative analysis of Ochratoxin A, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, and Aflatoxin G2 in cannabis biomass and cannabis derived products by HPLC with fluorescent detection. The samples analyzed with this consist of flower, resins, isolates, oils, vapes, edibles (chocolate, gummies), and beverages. Samples are extracted using an acetonitrile- water mixture with normal laboratory equipment and cleaned up using an immunoaffinity column prior to analysis. Baseline separation is achieved for the five analytes in less than 15 minutes with a gradient mobile phase. II. Review of the Method Only: II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. Yes 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. Yes 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. No, but the supporting documents include the definitions and apply them appropriately. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). Yes III. Review of Information in Support of the Method III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. Yes 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. No, the method authors didn't use reference materials because of the lack of cannabis reference materials. However, there are samples available through proficiency testing programs (not all matrices) that could be used instead of reference materials for accuracy and precision comparisons. 3. Is there information demonstrating that the method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability 2

should be modified. No, the supporting manuscripts published in Journal of AOAC provides the data to meet most of the SMPR method performance requirements specifications, but it is lacking data to support the linearity of the methods analytical range. IV. General Submission Package IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? No 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. Yes 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. Yes 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. Yes, the publication in Journal of AOAC clearly describes the method and addresses most of the requirements in the SMPR. 5. Based on the supporting information, what are the pros/strengths of the method? 1) Analytical method builds on previously approved AOAC methods for the inclusion of cannabis samples/products. 2) HPLC-FL method provides a baseline separation in less than 15 min for the five analytes. 3) Meets most of the "Method Performance Requirements" stipulated in the SMPR. 6. Based on the supporting information, what are the cons/weaknesses of the method? 1) Serial dilutions are used for the calibration curve instead of individual preparations from the original stock. 2) In lack of reference materials availability, no blind test samples were obtained from other sources such as PT for analysis to make a true accuracy comparison. 3) Samples are ground with a mortar and pestle but no requirements/recommendations are provided for minimum particle size. 4) Even though the recoveries meet the SMPR requirements, there is no supporting data to the optimization of the extraction procedure although the author mention they did the work. This is primarily concerning for the analysis of plant samples, where there consistently under 80% in the 2021 publication. Also, the recoveries for Aflatoxin G2 is generally lower than the others. Could better recoveries be obtained with a different solvents, longer extraction times, more extractions, etc.? 5) Vape, Resin, and Isolates were not evaluated at all three levels like the other matrix without an explanation. 6) The calibration ranges are provided but supporting data is not included such as the correlation coefficients. 7) Author should include chromatograms for the different samples analyzed in this study to demonstrate no matrix interferences. 7. Any general comments about the method? No

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