FAM Expert Review Panel (ERP) Training

AOAC INTERNATIONAL FOOD AUTHENTICITY METHODS (FAM) Expert Review Panel

Instructional Training Session (February 2022)

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

UNITED STATES dboyd@aoac.org

AOAC INTERNATIONAL FOOD AUTHENTICITY METHODS PROGRAM

Sharon Brunelle, Ph.D., Principal Consultant Brunelle Biotech Consulting AOAC Technical Consultant

Dr. Sharon Brunelle has worked with the AOAC RI and AOAC INTERNATIONAL since 2003 as a technical consultant coordinatingmicrobiology and chemistry method validation studies in the Performance Tested Methods SM (PTM) and Official Methods of

Analysis SM (OMA) programs. Brunelle also served as the technical lead for various contract-related and standards development projects within AOAC. She played a major role in developing guidelines for validation of biothreat agent methods and revising guidelines for validation of microbiology methods. Prior to AOAC, she worked for 10 years in diagnostics development and validation, both in foodmicrobiology and clinical oncology. Brunelle earned her bachelor’s degree in chemistry from the University of Delaware and a PhD in biochemistry from Brandeis University. Her postdoctoral work was in medical biochemistry at Rockefeller University and the Picower Institute for Medical Research.

OMA Training: Expert Review Panel for Food Authenticity Methods (FAM)

Sharon Brunelle, PhD AOAC Technical Consultant sharon@brunellebiotech.com

Official Methods of Analysis SM Program • Conformity assessment program for human and animal food, tissues, and environmental test methods • Microbiology (bacterial, fungal and viral) • Chemistry (drug residues, pesticide residues, allergens, toxins, proximate analysis, food fraud, etc.) • Species identification (meat, botanical) • Based on vote of the Expert Review Panel (ERP) – Final Action requires Official Methods Board (OMB) review and vote.

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AOAC Expert Review Panel Members • Attributes • Subject matter expert (with respect to analyte, technique, matrices) • Knowledge of AOAC processes and requirements • Ensure continuity of requirements from study to study • No conflict of interest • Responsibilities • Review Single Laboratory Validation (SLV) and Multi-laboratory Validation (MLV) protocols to assure claims are properly validated • Review validation study reports (SLV and MLV) • Review OMA methods • Provide recommendations of First and Final Action 3

Overview of the OMA Validation Process

Methods may be submitted: • In response to Call for Methods • From sole-source suppliers • From the PTM or R 2 Programs

SLV Manuscript, MLV Protocol, & Method Review

MLV Manuscript & Method Review

SLV Protocol

SLV Studies

Publication

MLV Study

ERP input Approve protocol

ERP input Recommend OMA Final Action to OMB

ERP input Determine OMA First Action; Determine requirements for OMA Final Action

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OMA SLV PROTOCOLS

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SLV and MLV Protocol Development

• Optional Consulting Service – Consulting application submitted by Method Developer (MD). – SLV and/or MLV Validation protocols are drafted by an AOAC Technical Consultant in collaboration with the MD. • Validation protocols based on • Method instructions – package insert/user manual/application note • Nature of the method (binary, multiplex binary, quantitative) • Scope of claim (matrices, concentration range) • Validation guidelines (AOAC OMA Appendices) • Relevant SMPR • Validation protocols reviewed and approved by Expert Review Panel and, if deemed necessary, by a Statistical Advisor.

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SLV Protocol Format

1. Title – includes name of method, mode of measurement, analyte(s), matrix(es), analytical technique, and type of study (SLV) 2. Authors - Sponsor(s)/Technical Consultant/Coordinating Laboratory 3. Introduction a. Method principle and description b. Validation overview and scope i. Relevant Guidelines ii. Relevant SMPR(s) iii. Scope of validation [matrix(es), test materials, reference materials]

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SLV Protocol Format

4. Good Laboratory Practices a. Randomizing/blind coding b. Safety precautions 5. Definitions 6. Reference Materials, reference standards, reference methods – identify and provide source or link. 7. Validation studies – identify where the studies will be performed and provide a specific study design and protocol for each study a. Selectivity b. Calibration model

c. Matrix Study d. Robustness

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SLV Protocol Appendices

• Method written in OMA style • OMA Report Template • Appropriate AOAC Statistical Workbook(s)

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Quantitative Chemistry Study Designs

Study

Design

Statistics

Criteria

Determine calibration model- response vs concentration of analyte

Regression - simple, order (linear, quadratic), weighting, etc. and residual analysis (plot residuals vs conc) Report positive and negative interferences

Calibration function should fit ƚŚĞ ĚĂƚĂ͖ ƌĞƐŝĚƵĂůƐ чϭϱй ;ůŽǁĞƐƚ ĐŽŶĐ чϮϬйͿ ǁŝƚŚ random pattern

Calibration

Calibration curve according to method instructions; consider solution vs. matrix-matched standards Test for interferences in the presence and absence of analyte All claimed matrixes tested in SLV; co-fortification if multianalyte method Plackett-Burman design - 7 parameters; 8 treatment combinations Full factorial design if 3 parameters varied Screening factorial design if 4-6 parameters varied 1 test material; 1-2 replicates per treatment combination 4- ϲ ĐŽŶĐĞŶƚƌĂƚŝŽŶƐ ;ŝŶĐů ϬͿ 5 replicates per concentration

Determined by SMEs

Selectivity

Determined by SMEs; suggested recovery and precision criteria in OMA Appendix K

Recovery/bias, LOD/LOQ, s r

Matrix Study

Regression analysis per matrix Intermediate precision*

Significant findings do not mean method fails. Parameter settings that affect method performance require warnings or limitations in method.

Multiple regression analysis, ANOVA, or generalized linear model analysis

Robustness

*May be required by some communities

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Binary (Qualitative) Chemistry Study Designs - OMA Appendices K & N

Study

Design

Statistics

Criteria

Report positive and negative interferences Determined by SMEs

Test for interferences in the presence and absence of analyte

Selectivity

ĚWK ϵϱй / ŵƵƐƚ ŝŶĐůƵĚĞ Ϭ ŝĨ ŵĞƚŚŽĚ comparison

All claimed matrixes tested in SLV; co-fortification if multi-analyte method

Matrix Study

POD

ϰ ĐŽŶĐĞŶƚƌĂƚŝŽŶƐ ;ŝŶĐů ϬͿ – 2 conc. yielding fractional positive results

ĞƚĞƌŵŝŶĞ ϵϬй WK ǁŝƚŚ ϵϱй ĐŽŶĨŝĚĞŶĐĞ ŝĨ ŶŽƚ method comparison Significant findings do not mean method fails. Parameter settings that affect method performance require warnings or limitations in method.

ϮϬ - ϯϬ ƌĞƉůŝĐĂƚĞƐ ƉĞƌ ĐŽŶĐĞŶƚƌĂƚŝŽŶ

Full factorial design if 3 parameters varied

^ĐƌĞĞŶŝŶŐ ĨĂĐƚŽƌŝĂů ĚĞƐŝŐŶ ŝĨ шϰ ƉĂƌĂŵĞƚĞƌƐ ǀĂƌŝĞĚ

Generalized linear model

Robustness

1 material at fractional positive level (2-4 positives/5 replicates

5 replicates per treatment combination

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Review of SLV Validation Protocols

• Claims – are method claims clearly stated? • Validation Guidance – are appropriate Guidelines and/or SMPRs cited? • Selectivity – Appropriate molecules (similar in structure or activity to the analyte) for potential interference – Interference tested in the presence and absence of analyte – ƉƉƌŽƉƌŝĂƚĞ ĐŽŶĐĞŶƚƌĂƚŝŽŶ;ƐͿ ƚĞƐƚĞĚ ;ŐĞŶĞƌĂůůLJ ϭϬϬdž ŚŝŐŚĞƌ ƚŚĂŶ ĂŶĂůLJƚĞͿ • Calibration – Appropriate number and range of calibration standards – Solution or Matrix-matched calibration standards • Matrix effect? – Residual plot and residual size included to support calibration model

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Review of SLV Validation Protocols

• Matrix study

• Full validation of all claimed matrices • Quantitative methods: Accuracy, repeatability precision, bias, LOD/LOQ, intermediate precision • Qualitative methods: POD, bias • Proper study design (qualitative/quantitative) • Concentration levels • Replicates • Correct choice of challenge materials to represent the matrixes and concentration range of target parameter • Proper fortification protocols (if applicable) • Proper choice of reference method(s), reference materials and/or reference standards for each matrix to assess bias and/or accuracy • Correct test portion mass/volume for each material

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Review of SLV Validation Protocols

• Matrix Study – Data Analysis • Are all appropriate method performance parameters identified and defined? • Are equations correctly presented? • Is an appropriate and current AOAC Statistical Workbook available and cited? • Are LOD/LOQ estimated appropriately for this situation? Consider technology and availability of known negative matrixes. • Will estimated LOQ be validated for each matrix? • Are acceptance criteria from SMPR clearly stated, if applicable? • Robustness • Parameters varied are appropriate to test robustness of the method • Appropriate experimental design (factorial or fractional factorial) • Appropriate data analysis

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OMA MLV PROTOCOLS

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MLV Protocol Format

1. Title – includes name of method, mode of measurement, analyte(s), matrix(es), analytical technique, and type of study (SLV) 2. Authors - Sponsor(s)/Technical Consultant/Coordinating Laboratory 3. Introduction a. Method principle, description, scope, and brief summary of SLV results b. MLV Validation overview and scope i. Relevant Guidelines ii. Relevant SMPR(s) iii. Scope of validation [matrix(es), test materials, reference materials]

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MLV Protocol Format

4. Good Laboratory Practices a. Randomizing/blind coding b. Safety precautions 5. Definitions 6. Reference Materials, reference standards, reference methods – identify and provide source or link. 7. Validation study a. Potential collaborators b. Study design c. Preparation of test materials, packaging and shipping of test portions, storage of test portions upon receipt d. Reporting raw data – how and to whom will raw data be submitted? e. Analyzing raw data – outlier treatment, statistical analyses, AOAC statistical workbook

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MLV Protocol Appendices

• Method written in OMA style • SLV Report • Instructions to Collaborators • Data Reporting Form • Appropriate AOAC Statistical Workbook • OMA Report Template

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OMA MLV Study Designs

Method Type # Valid Data Sets Study Design per Collaborator

Statistics

Recovery, bias, LOD, LOQ, s r , s R

Quantitative

шϴ

шϱ DĂƚĞƌŝĂůƐ 2 Replicates per material 1 Matrix 12 Replicates High 12 Replicates Low (fractional pos.*) 12 Replicates Non-spiked

Qualitative

шϭϬ

LPOD s r ǁŝƚŚ ϵϱй / s R ǁŝƚŚ ϵϱй / s L If method comparison: dLPOD н / ; / ŵƵƐƚ ŝŶĐůƵĚĞ ͞Ϭ͟ )

Ύ&ƌĂĐƚŝŽŶĂů ƉŽƐŝƚŝǀĞƐ ĚĞĨŝŶĞĚ ĂƐ >WK с Ϭ͘ϮϬ - Ϭ͘ϴϬ Updates under consideration for qualitative MLV statistics: /ŶƚƌĂĐůĂƐƐ ŽƌƌĞůĂƚŝŽŶ ŽĞĨĨŝĐŝĞŶƚ ;/ Ϳ ƚŽ ĂƐƐĞƐƐ ŚŽŵŽŐĞŶĞŝƚLJ ŽĨ ĐŽůůĂďŽƌĂƚŽƌƐ ;ϵϬй / ŽŶ / фϬ͘ϰͿ͖ ƋƵŝǀĂůĞŶĐĞ ĂƐ ϵϬй / ŽŶ dLPOD CP (- Ϭ͘Ϯ͕ Ϭ͘ϮͿ͕ ϵϬй / ŽŶ dLPOD C (- Ϭ͘Ϯ͕ ϭ͘ϬͿ͘

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Review of MLV Validation Protocols

• Collaborators • YƵĂůŝƚĂƚŝǀĞ ŵĞƚŚŽĚƐ͗ шϭϬ ǀĂůŝĚ ĚĂƚĂ ƐĞƚƐ͕ ƌĞĐŽŵŵĞŶĚ ϭϮ -15 collaborators • YƵĂŶƚŝƚĂƚŝǀĞ ŵĞƚŚŽĚƐ͗ шϴ ǀĂůŝĚ ĚĂƚĂ ƐĞƚƐ͕ ƌĞĐŽŵŵĞŶĚ ϭϬ -12 collaborators • No more than 3 collaborators at one site (must work independently!) • At least 5 sites • Method training provided (in-person or virtual) • Recommend practice samples to qualify collaborators and familiarize them with the method • Coordinating Laboratory can also be collaborator (separate personnel preparing materials and analyzing sample set)

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Review of MLV Validation Protocols

• Study Materials

• шϱ DĂƚĞƌŝĂůƐ ĂƐ ďůŝŶĚ ĚƵƉůŝĐĂƚĞƐ

• Material = Matrix/Analyte/Concentration combination • Do materials cover the variety of the claim? • Is the most challenging material(s) from the SLV included in the MLV?

• Homogenization and portioning performed by Coordinating Laboratory • Portions are randomized and blind coded (unique identifiers for each collaborator) • Shipping to collaborators • Sample stability – Temperature control and/or monitoring needed? • Overnight shipment • Shipping follows IATA regulations (dangerous goods) if applicable? • Itemized packing list provided for each Collaborator? • Study Reagents/Supplies • Are reagents/supplies provided by the Coordinating Laboratory and those required to be sourced by the Collaborator clearly delineated in the Instructions to Collaborators? 21

Review of MLV Validation Protocols

• Study Timeline • Is the study timeline clearly presented and communicated in the protocol and in the Instructions to Collaborators? • Shipping date(s) • Receipt date(s) • Training (can be a virtual meeting to review method) • Practice sample analysis • Review of practice sample data and possible re-training to address issues • Study sample analysis • Deadline for submitting raw data

• Do all collaborators need to start on the same day or is a time range appropriate within which the test portions should be analyzed, and data reported?

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Review of MLV Validation Protocols • Data Submission – a data report form is highly recommended to ensure capture of all relevant information for all analyses • Name of laboratory • Name of analyst

• Date(s) of analysis • Equipment used • Critical reagents/supplies documented (catalog #, lot#) • Calibration curve(s) • Raw data • Calculated results • Critical parameters (e.g., dilution factor) • Protocol or method deviations • Data QC-checked?

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Review of MLV Validation Protocols

• Data Analysis • Are all appropriate method performance parameters identified and defined? • Are equations correctly presented? • Is an appropriate and current AOAC Statistical Workbook available and cited? • Are acceptance criteria from SMPR clearly stated, if applicable?

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OMA METHODS

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OMA Method Format

Title - Method number, method name, technology, First Action year, Final Action year Applicability statement

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OMA Method Format

Caution – precautions, possible hazards, safe handling Reference to data tables supporting method reproducibility

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A. Principle – description of technology and method OMA Method Format

Etc.

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OMA Method Format

B. Apparatus – list of required equipment and sources; trademark symbols may be used on first citing only C. Chemicals and Reagents – list of chemicals and reagents and their sources Formatting: • Major headings labeled with upper case letters; bold font • Lists begin with lower case letters in parenthesis, bold font, indent first line • Sub-lists labeled with numeral in parenthesis; italic font, indent first line

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OMA Method Format

D. Preparation of

Reagents – how to prepare reagents, calibration standards, mobile phases, etc., as needed

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OMA Method Format

E. General Preparation – set up of apparatus, general instructions, as needed F. Sample Preparation – comminution of laboratory sample, selection and extraction of test portion; may have different instructions for different matrix types

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OMA Method Format

G. Determination – how to perform the analytical analysis H. System Suitability – checks to assess proper analytical system performance I. Calculations – instructions for calibration curve determination and results determination including equations.

Etc.

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OMA Method Format Note for table and figure numbering : Tables and Figures within the method should be numbered ĂƐ ĨŽůůŽǁƐ͗ ϮϬϮϮ͘dždž ͕ ϮϬϮϮ͘dždž ͕ ŝŶĐůƵĚŝŶŐ ĚĂƚĂ supporting the reproducibility of the method (First Action for microbiology, Final Action for chemistry)

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Review of OMA Method

• Are sample preparation instructions clear? • Are analysis and interpretation instructions clear? • Are calculations clearly presented, and correct? • Are instructions consistent with other MD publications (e.g., package insert or application note)? • Are safety precautions clearly stated? • Are claims consistent with the proposed protocol?

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AOAC RI Package Insert/User Manual/ Application Note Requirements

As applicable:

– Intended User - Specify the intended user – Environmental Factors - Identify the environment that the test should be conducted – Applicability Statement - Identify the analyte, mode of measurement and matrices for which the method has been validated – Limitations – Disclose all known limitations or interferences – System Suitability – Requirements for determining valid equipment and component performance (performance qualification). – Instructions - Include complete instructions from sample preparation to data interpretation – Shelf Life – The shelf life and storage conditions of the test kit and its components – Detection Limit and Limit of Quantitation - Express in concentration (parts per million, percent, mg/kg, CFU/g, etc.) the limits of detection and quantitation – Precautions - Provide warnings of safety concerns, disposal instructions, and potentially hazardous steps or components. – Technical Assistance - Provide information (email, internet, telephone and FAX numbers) where the user can obtain technical assistance. 35

OMA REPORTS

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OMA Report Format

Manuscript should be double-spaced in 12 pt Times New Roman. If referring to an AOAC method number in the text (or reference list) the number should be bold: AOAC Method 2015.01 . Title Validation of the Test Method/Modification for Detection/Determination/Enumeration of Analyte in Matrix(es): First/Final Action 20xx.xx Authors’ names Affiliations Corresponding author’s email: ORCID Number: All authors are required to submit their ORCID number. Please list the author’s name and their number here. Visit www.orcid.org to register for an ORCID number.

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OMA Report Format

Abstract

Note: No more than 300 words. Abstracts should be organized by background, objective, methods, results, conclusions, highlights (with section titles in bold as shown above). No indent for the abstract only. Do not cite tables, figures, or references in the Abstract. Spell out or define terms and follow with an abbreviation/acronym in parentheses if the term is used more than once in the Abstract . Do not include the abbreviation/acronym if the term is not used more than once. Do not include any information that is not part of the study reported (e.g., future plans or suggestions). ( Note : Acronyms/abbreviations, definitions, names of bacteria, etc., must be identified/spelled out in the body of the paper even though they were written out or defined in the Abstract.) Should contain a brief description of the method, matrixes tested and brief summary of results. dŚĞ ƚĞƌŵ ͞ŵĂƚƌŝdžĞƐ͟ ŝƐ ƵƐĞĚ ŝŶ JAOAC publications, instead of matrices.

Background: Objective: Methods: Results: Conclusions: Highlights:

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OMA Report Format

Introduction

• Background information on the analyte and relation to food safety. Why is a new validated method needed? What are the advantages of the new method? • Cite references in numerical order with number in parenthesis in line with text (not superscript) • Tables and Figures outside of the OMA Method should be numbered consecutively (Table 1, Table 2, etc.). Title Tables and Figures using a period after the Table or Figure number (Table 1. Test materials used for the SLV study) • SI Units: use SI units such as g, L, mL, min, μ g/kg (not ppm or μ g kg -1 ) • A list of standard acronyms and initialization can be found at ŚƚƚƉ͗ͬͬǁǁǁ͘ĞŽŵĂ͘ĂŽĂĐ͘ŽƌŐͬǁĞďйϮϬĚĞĨŝŶŝƚŝŽŶйϮϬŽĨйϮϬƚĞƌŵƐйϮϬĂŶĚйϮϬĞdžƉ ůĂŶĂƚŽƌLJйϮϬŶŽƚĞƐ͘ƉĚĨ .

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OMA Report Format

Single Laboratory Validation Study Selectivity Calibration Model Matrix Study Robustness Multilaboratory Validation Study Study Design Preparation of Test Portions Test Portion Distribution Test Portion Analysis Statistical Analysis [Method in OMA Style]

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OMA Report Format

Results (SLV) Selectivity Calibration Model Matrix Study Robustness Results (MLV) Raw Data Statistical Analysis

Discussion Conclusion Acknowledgements Conflict of Interest (e.g., ‘All authors declare no conflict of interest.’ ) Funding (if applicable) References (list numerically exactly as examples: https://academic.oup.com/jaoac/pages/General_Instructions ) Example report: ŚƚƚƉƐ͗ͬͬĂĐĂĚĞŵŝĐ͘ŽƵƉ͘ĐŽŵͬũĂŽĂĐͬĂƌƚŝĐůĞͬϭϬϯͬϲͬϭϱϯϰͬϱϴϲϲϳϬϬ

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OMA SLV Review

An OMA SLV submission may originate from: 1. A Method Developer a. A protocol may or may not have been submitted for review by the ERP 2. A Method Developer who used the OMA Consulting Service a. A protocol was reviewed by the ERP 3. A Method Developer with a method certification from the AOAC Research Institute Performance Tested Methods SM (PTM) or Reviewed & Recognized SM (R 2 ) Program a. A protocol was reviewed, possibly by one ERP member acting as AOAC Volunteer Expert for the AOACRI b. A validation study report was reviewed and approved by the AOAC Volunteer Expert and 2 Expert Reviewers. PTM and R 2 validations include data from 1 IL.

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OMA SLV Review

ERP Method Review Form: Preliminary Questions • Is the Validation Study Report in a format acceptable to AOAC? • 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 tables and figures 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? • Does the method contain adequate safety precaution references and/or statements? ERP Method Review Form: I. Summary of the Method II. Review of Method Only 1. Does the applicability of the method support the applicability of the SMPR? If no, explain. 2. Does the analytical technique(s) used in the method meet the SMPR? If no, explain. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, explain. 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, suggest wording. OMA SLV Review 43

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OMA SLV Review

ERP Method Review Form: III. Review of Information in Support of the Method

1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscript, method studies, etc.)? If no, explain. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If no, explain. 3. Is there information demonstrating the method performs within the SMPR Method Performance Requirements table specifications for all analytes in the SMPR applicability statement? If no, explain.

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OMA SLV Review

ERP Method Review Form: 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? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If no, indicate if there is a need for such tests or controls and which ones. 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, explain.

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OMA SLV Review

ERP Method Review Form: IV. General Submission Package (cont.)

4. Based on the supporting information, is the method written clearly and concisely? If no, specify revisions needed. 5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method? 7. Any general comments about 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 provide rationale.

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FOOD AUTHENTICITY METHOD VALIDATIONS

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FAM Validation Resources

SMPRs: https://www.aoac.org/resources/?topic=&type=SMPRs&key=

Commodity Adulterant(s)

Targeted Testing ϮϬϮϬ͘ϬϬϵ ϮϬϮϬ͘ϬϭϬ ϮϬϮϬ͘Ϭϭϭ

Nontargeted Testing

Honey

C3 and C4 Sugars

ϮϬϮϬ͘ϬϬϲ ϮϬϮϬ͘ϬϬϳ ϮϬϮϬ͘ϬϬϴ

EV olive oil

Veg. and low quality olive oils

Milk

Formaline/formaldehyde, starch, soy

Saffron

[Numerous] [Numerous] [Numerous]

Draft Draft Draft

Draft Draft Draft

Turmeric

Vanilla

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FAM Validation Resources

OMA Appendices: http://www.eoma.aoac.org/appendices.asp Appendix D : Guidelines for Collaborative Study Procedures Appendix G : Procedures and Guidelines for the Use of AOAC Voluntary Consensus Standards to Evaluate Characteristics of a Method of Analysis Appendix K : Guidelines for Dietary Supplements and Botanicals Appendix N : ISPAM Guidelines for Validation of Qualitative Binary Chemistry Methods

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FAM SLV Validations - TT

Analytical Techniques • Any method able to detect, identify, and quantitate target adulterants will be considered. – Can a method be validated for detection only? Samples • Authentic products must be accompanied by appropriate documentation or testament of authenticity. • Authentic products should include botanical, regional, and seasonal variations as appropriate. • Whole liquid bovine milk types include raw, pasteurized, UHT, and sterilized. Method claim based on scope of validation.

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FAM SLV Validations - TT

Performance Parameters – Quantitative Methods • Analytical range – quantitation range for each targeted adulterant • LOD/LOQ – lowest concentration that can be detected (LOD) or quantitated with acceptable precision (LOQ) – there are many ways to estimate LOD and LOQ – estimated LOQ (LOQ est ) should be validated for each matrix • Recovery – candidate method result (or mean) divided by the known fortified concentration • Accuracy (Trueness or bias) – the difference between the candidate method result (or mean) and the known concentration • Repeatability precision (RSD r ) – relative standard deviation under repeatability conditions • Reproducibility precision (RSD R ) - relative standard deviation under reproducibility conditions • HorRat values – RSD r /PRSD R and RSD R /PRSD R

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FAM SLV Validations - TT

Performance Parameters – Qualitative Methods • Probability of Identification Model (OMA Appendix K Part II) – POI = x/n, where x is number of ‘adulteration not detected’ results and n is the number of test portions – &ŽƌƚŝĨLJ ŵĂƚƌŝdž ǁŝƚŚ ĂĚƵůƚĞƌĂŶƚ Ăƚ ϭϬй͕ ǁŚŝĐŚ ŝƐ ƚŚĞ >KY ƐƉĞĐŝĨŝĞĚ ŝŶ ^DWZ ;^ƉĞĐŝĨŝĞĚ /ŶĨĞƌŝŽƌ Test Material, SITM) – &ŽƌƚŝĨLJ ŵĂƚƌŝdž ǁŝƚŚ ĂĚƵůƚĞƌĂŶƚ Ăƚ >K с >KYͬϯ с ϯ͘ϯй ;^ƉĞĐŝĨŝĞĚ ^ƵƉĞƌŝŽƌ dĞƐƚ DĂƚĞƌŝĂů͕ SSTM) – &ƌŽŵ ŶŶĞdž ͕ ĂƐƐƵŵŝŶŐ ŵĂdž͘ ϭϬй ĞƌƌŽƌ ĂŶĚ ĂůůŽǁŝŶŐ ϭ ĞƌƌŽŶĞŽƵƐ ƌĞƐƵůƚ͕ ƚŚĞ ƐĂŵƉůĞ ƐŝnjĞ ŝƐ 48 replicates – Test:

• 48 replicate test portions SITM • 48 replicate test portions SSTM • ϯϬ ƌĞƉůŝĐĂƚĞ ƚĞƐƚ ƉŽƌƚŝŽŶƐ ĐŽŶƚƌŽů ŵĂƚƌŝdž ;ŶŽƚ ĂĚƵůƚĞƌĂƚĞĚͿ • ZĞƉŽƌƚ WK/ ǁŝƚŚ ϵϱй ĐŽŶĨŝĚĞŶĐĞ ŝŶƚĞƌǀĂů ĨŽƌ ^/dD͕ ^^dD͕ ĂŶĚ ĐŽŶƚƌŽů ŵĂƚƌŝdž

• Probability of Detection – ůƚĞƌŶĂƚŝǀĞůLJ͕ ƚĞƐƚ ϲ ĐŽŶĐĞŶƚƌĂƚŝŽŶƐ ŽǀĞƌ ƚŚĞ ƌĂŶŐĞ Ϭ ƚŽ ϭϬй ĂŶĚ ƉůŽƚ WK ǀƐ й ĂĚƵůƚĞƌĂƚŝŽŶ • ŝŶĐůƵĚĞ Ăƚ ůĞĂƐƚ ϯϬ ƌĞƉůŝĐĂƚĞƐ ƉĞƌ ĐŽŶĐĞŶƚƌĂƚŝŽŶ • ĚĞƚĞƌŵŝŶĞ ůŽǁĞƐƚ ĐŽŶĐĞŶƚƌĂƚŝŽŶ ƚŚĂƚ ƉƌŽǀŝĚĞƐ ϵϱй ĚĞƚĞĐƚŝŽŶ ŽĨ ĂĚƵůƚĞƌĂƚŝŽŶ ǁŝƚŚ ϵϱй ĐŽŶĨŝĚĞŶĐĞ 53

FAM SLV Validations - TT

Robustness Testing – Method-specific – Factorial or fractional factorial design

Selectivity Testing? • FAM SMPRs do not provide guidance on selectivity testing – Test a variety of targeted adulterants • Ğ͘Ő͕͘ ͞ŽƚŚĞƌ ǀĞŐĞƚĂďůĞ ŽŝůƐ͟ ĨŽƌ ^DWZ ϮϬϮϬ͘ϬϭϬ ĐŽƵůĚ ŝŶĐůƵĚĞ ĐĂŶŽůĂ͕ ĂǀŽĐĂĚŽ͕ ĐŽƌŶ͕ ĐŽĐŽŶƵƚ͕ cottonseed, palm, safflower, sesame, soybean, sunflower, peanut, hemp, etc. – Is there a need to test substances outside the list of targeted potential adulterants?

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FAM SLV Validations - NTT

Analytical Techniques • Binary methods that generate a baseline fingerprint of authentication to which test materials are compared. Samples • Authentic products must be accompanied by appropriate documentation or testament of authenticity. • Authentic products should include botanical, regional, and seasonal variations as appropriate. • Whole liquid bovine milk types include raw, pasteurized, UHT, and sterilized. Method claim based on scope of validation. • Validation materials should be sourced separately from those used to create the database (different source or lot number).

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FAM SLV Validations - NTT

Performance Testing – For each matrix, test each potential adulterant at the concentration specified in the SMPR • ϯϬ ƌĞƉůŝĐĂƚĞƐ ŽĨ ĞĂĐŚ ĂĚƵůƚĞƌĂƚĞĚ ƐĂŵƉůĞ • ϯϬ ƌĞƉůŝĐĂƚĞƐ ŽĨ ĂƵƚŚĞŶƚŝĐ ƐĂŵƉůĞ • ϭϬϬй ĐŽŶĐŽƌĚĂŶĐĞ ǁŝƚŚ ŬŶŽǁŶ ĂĚƵůƚĞƌĂƚŝŽŶ ƐƚĂƚƵƐ ƌĞƋƵŝƌĞĚ

Robustness Testing

– Method-dependent – factorial or fractional factorial design

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QUESTIONS?

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