AOAC® Official Methods^SM Validation Program

AOAC INTERNATIONAL is a unique, nonprofit scientific organization whose primary purpose is to serve the needs of government, industry, and academic laboratories for analytical methods and quality measurement systems. The AOAC® Official Methods^sm Program is designed to provide methods of analysis with known performance characteristics, such as accuracy, precision, sensitivity, range, specificity, limit of measurement, and similar attributes. A prerequisite of AOAC adoption is validation through interlaboratory collaborative study in independent laboratories under identical conditions. Such validated methods can then be used with confidence by regulatory agencies, regulated industry, product testing laboratories, and academic institutions. The methods are used to determine compliance with government regulations, to maintain quality control and process requirements, to set and evaluate compliance with terms of procurement contracts, to conduct national and international trade, and to support research.

The actual work is done worldwide by appointed volunteers in their professional capacities as scientists of federal, state, provincial, and municipal laboratories; academic and experiment station laboratories; and commercial laboratories. These volunteers contribute time, expertise, and laboratory capability to participate as researchers, methods collaborators, committee members, and advisors.

AOAC INTERNATIONAL has over a century of experience in using the interlaboratory collaborative study as a means of determining the performance characteristics of a method for both general and regulatory use. AOAC’s major contribution to analytical science has been to bring the interlaboratory collaborative study technique to a high degree of perfection, and to encourage other methods organizations to harmonize their programs with the AOAC procedure. As stated in the U.S. Code of Federal Regulations (Title 21), it is the policy of the U.S. Food and Drug Administration in its enforcement programs to use the methods of analysis of AOAC INTERNATIONAL as published in the latest edition (18th Ed., 2005) of their publication Official Methods of Analysis of AOAC INTERNATIONAL. In addition, in the U.S. Code of Federal Regulations (Title 9), Animal and Animal Products, Official Methods of Analysis of AOAC INTERNATIONAL (15th Ed., 1990), is incorporated by reference with the approval of the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR Part 51.

METHOD SUBMISSION

Several mechanisms exist for submitting methods to AOAC: (1) a government agency or an organization may enter into a contract with AOAC for the validation of specific methodology or provide continuous infrastructure support for the review and approval of methods in a particular area of interest; (2) a community may be formed comprised of stakeholders from government, industry, and/or academia in a particular area of interest who need validated methods and submit best and most needed methods for AOAC review and approval; (3) a company or organization that, for example, has a proprietary product and has an interest in obtaining an economic advantage through the AOAC approval of their method may submit its methods for review and approval, together with a submission fee.

Communities

Methods may be submitted by AOAC analytical communities. Communities bring together analytical scientists in a specific area who share a commitment to consolidate efforts to prioritize method needs, establish performance criteria, gather and evaluate existing methods, help seek funding, and support validation work for methods that are fit-for-purpose. Communities may be able to secure collaborative funding from industry and government in support of much needed methodology. Examples of AOAC analytical communities various stages of development include, but are not limited to, Agricultural Materials, Dietary Supplements, Food Allergens, Homeland Security, and Marine and Freshwater Toxins.

For more on AOAC’s various communities, visit our Web site at www.aoac.org and click on “AOAC Analytical Communities.”

Contracts and Infrastructure Support

An interested party, usually a government agency, in need of validated methods may build a contractual relationship with AOAC.

For more information about government and industry participation, contact Anita Mishra at amishra@aoac.org or Tel: +1-301-924-7077 ext. 131.

Individual Company or Organization

Methods may be submitted by an individual company or organization that has, for example, a proprietary product and wants to have their method(s) approved by AOAC.

METHOD VALIDATION PROCESS

Method Development and In-House Study

An AOAC-sponsored validation study begins with the appointment of a Study Director, the individual scientist who is responsible for organizing the method study.

He or she selects, develops, or adopts a method to be studied. For a microbiology method, a precollaborative study is required, to be conducted according to AOAC guidelines. In the case of a chemistry method, the Study Director develops the required in-house validation data and collaborative study protocol (design) in accordance with AOAC guidelines.

The recommended study protocol and in-house validation data are reviewed by the General Referee, Committee Statistics and Safety Advisors, up to 2 Methods Committee members, and the Methods Committee Chair. Once an agreement on the study protocol is reached, the Study Director can begin the interlaboratory collaborative study.

Validation Through Collaborative Study

The Study Director recruits collaborators in laboratories with experience in the type of analysis required in the proposed method. For quantitative methods, AOAC INTERNATIONAL requires valid data from no fewer than 8 laboratories, each analyzing a minimum of 5 materials, as blind duplicates or Youden pairs. For qualitative methods, the minimum criteria are 15 laboratories reporting 2 analyte levels per matrix, 5 replicates per level, and 5 negative controls per matrix.

The Study Director prepares the test materials and, if required, other materials to be supplied to collaborators, such as reference materials, column packings, or monoclonal antibodies, and ships them to the cooperating laboratories along with the method, instructions for conducting the study, and reporting forms.

Collaborators are expected to conduct the test exactly as instructed and according to the method, with no deviations, and return results within the time frame agreed.

Expert Review

The Study Director compiles the data, evaluates the results, and writes the collaborative study report, in accordance with AOAC guidelines. Statistical treatment of the data is considered essential in a rigorous evaluation of the method, and AOAC INTERNATIONAL provides manuals, statistical software, and expert consultation to aid the Study Director.

The report is submitted to the General Referee and Statistics Advisor and then to the Methods Committee and 2 Official Methods Board members for technical review. Methods acceptable through these review levels are then approved for adoption as First Action AOAC® Official Methods^sm.

Adoption of First Action AOAC® Official Methods^sm

A Methods Committee reviews the submitted collaborative study reports, comments, and associated documentation to ensure adherence to the technical review process. Advance notices of the methods to be considered for First Action are published in the Referee section of AOAC’s magazine, Inside Laboratory Management, and on the AOAC Web site.

Method actions taken by the Methods Committees are published in the Referee section of AOAC’s magazine, Inside Laboratory Management. The complete text of newly adopted AOAC® Official Methods^sm and the reports or summaries of the interlaboratory collaborative studies are published in the Journal of AOAC INTERNATIONAL. The adopted methods are added to the compendium, Official Methods of Analysis of AOAC INTERNATIONAL.

Adoption of Final Action AOAC® Official Methods^sm

First Action AOAC® Official Methods^sm are eligible for Final Action status after they have been available in the literature for at least 2 years. If the Association has not received any information as to significant problems in the performance of the method, the General Referee recommends adoption of the method as Final Action, and the method is listed in the Referee section of AOAC’s magazine, Inside Laboratory Management, and on the AOAC Web site so interested parties may submit comments and data if desired.

A ballot of methods recommended by the General Referees and Methods Committees for Final Action is submitted to the Official Methods Board who votes on the acceptance of the methods as Final Action. Notices of the methods adopted as Final Action AOAC® Official Methods^sm are published in the Referee section of AOAC’s magazine, Inside Laboratory Management, and on the AOAC Web site.

Actions Affecting AOAC® Official Methods^sm

Methods can be repealed, in which case they lose their official status. Methods are repealed through recommendations initiated by the General Referee and approved by the Methods Committee and Official Methods Board. Notification of the recommendation is made through publication in the Referee section of AOAC’s magazine, Inside Laboratory Management, so interested parties may submit comments and data on the proposed action.

Adoption of Methods Not Sponsored by AOAC INTERNATIONAL

Methods from other organizations that follow the AOAC harmonized protocol and are formatted in AOAC style may be submitted for AOAC review and adoption as AOAC® Official Methods^sm. Such methods enter the AOAC process at the point of technical review of the completed collaborative study.

Modifications to AOAC® Official Methods^sm

When it is necessary to make a modification in an existing , AOAC® Official Methods^sm, the procedure and extent of validation depend on the extent of the revision, whether editorial, minor, or substantive. These determinations are made by the General Referee and Methods Committee.

Appeals Process

All requests for review of AOAC® Official Methods^sm or method action must be submitted in writing. Each request is reviewed similarly to a method; the Official Methods Board then acts on the recommendation of the General Referee and Methods Committee.

COMMITTEE ORGANIZATION

Appointments

The AOAC® Official Methods^sm Program is administered by volunteer technical experts appointed by the AOAC President or a designee. Volunteers are generally appointed for 3-year terms, and each position has stated appointment requirements, duties, and responsibilities. Persons appointed as Official Methods Board and Methods Committee members and General Referees must be members of AOAC INTERNATIONAL because of their role in review and recommendation or adoption of AOAC® Official Methods^sm.

Official Methods Board

The Official Methods Board consists of the Chairs of the 11 Methods Committees plus a Board Chair and Vice Chair. The Board recommends, implements, and promotes uniform policies for the consideration and adoption of AOAC® Official Methods^sm, including statistical and safety requirements; grants Final Action status for First Action AOAC® Official Methods^sm, addresses requests for action; and resolves disputes in the AOAC® Official Methods^sm Program in accordance with established policies.

Methods Committees

The 11 Methods Committees each have 7–11 members plus a Chair, Secretary, and a committee Statistician and Safety Advisor. Each of the Methods Committees guides and supervises the development and validation of analytical methods for the identification and/or quantitation of analytes from a variety of matrixes; reviews protocols for interlaboratory studies; reviews completed studies and methods; approves methods for First Action; recommends actions on revision, repeal status; recommends scientists for appointment as General Referees; and recommends new General Referee topic areas for study.

General Referees

General Referees are organized along topic lines under appropriate Methods Committees. The General Referee is responsible for a broad area of study (e.g., Fertilizers; Fruits and Fruit Products; Drugs in Feeds; Mycotoxins) and coordinates and guides the activities of a number of Study Directors working on specific methods within the broad topic area. Each General Referee works with the Study Directors on methods development concepts; reviews the reports of Study Directors; recommends appropriate action on methods; and prepares an annual report to the Methods Committee on scientific issues in the designated area.

Study Directors

Study Directors are organized along topic lines under appropriate General Referees and Methods Committees. A Study Director conducts the interlaboratory study of a specific method in a topic area (e.g., a specific drug; a specific food additive; a specific feed component). A Study Director selects test methodology; develops in-house validation data; develops a protocol for the interlaboratory collaborative validation of the method; evaluates the completed study; recommends methods for adoption as First Action Official Methods^sm; and recommends appropriate First Action methods for adoption as Final Action AOAC® Official Methods^sm. Study Directors are required to submit an annual status report on the topic to the General Referee.

Topic Advisors and Method Advisors

Topic Advisors are responsible for assisting the General Referee in an assigned subject area. They research their topic area and provide recommendations for new methods that are needed. They provide guidance to Study Directors in designing a collaborative study.

Method Advisors serve as experts on specific methods. They answer technical inquiries about the method and provide recommendations for method modifications based on feedback by method users.

Collaborators

Any scientist experienced in analysis and qualified in the subject matter may collaborate in the study of a method. Collaborators are chosen by the organizer of the collaborative study from laboratories with an interest in the method, including regulatory agencies, industry, commercial laboratories, and universities. A collaborator is expected to analyze materials at times indicated, according to a protocol submitted by the Study Director; follow the method exactly (this is critical); report any unavoidable deviation; perform only the number of determinations requested; and supply raw data, graphs, recorder tracings, photographs, or other documentation.

Safety Committee

Safety Committee members have an interest in the safety and health aspects of the validation and use of analytical methods. The Committee promotes an awareness of safety and health matters within the AOAC membership; serves as a pool of expertise for the AOAC membership in regard to safety matters; submits safety awareness information for publication in Inside Laboratory Management; and establishes liaisons with other professional organizations to exchange safety information.

Statistics Committee

Statistics Committee members provide advice on statistical criteria and analysis of validation studies. The Committee develops and recommends harmonized statistical guidelines; encourages greater use of standardized statistical techniques; advises the Official Methods Board on statistical matters; educates AOAC volunteers in proper application of statistical techniques; and encourages greater use of statistical techniques.

Volunteer Participation and Conflicts of Interest

Members of committees, advisors, and referees may be chosen who, because they are experts in the subject area, may have conflicts or apparent conflicts in the performance of their duties. While this will not necessarily disqualify a volunteer from carrying out his or her duties, it is the sense of AOAC INTERNATIONAL that conflicts of interest or even the appearance of conflicts of interest should be avoided. Where it is not practical to eliminate all conflicts, AOAC policy states that these conflicts must be disclosed. All volunteers appointed in the AOAC® Official Methods^sm Program are required to sign a form accepting their appointment and agreeing to the provisions of the conflict of interest policy.

PRELIMINARY WORK

Purpose and Scope of the Method: The purpose and scope of the method must be decided. A method must be chosen and demonstrated to apply to the matrixes and concentration ranges of interest.

Optimization of New or Available Method: A collaborative study should not be conducted with a nonoptimized method. As much experimentation must be done within a single laboratory as possible with respect to optimization, ruggedness, bias, concentration–response curves, and interferences; the critical steps and variables should be determined and the need for their control emphasized.

Description of the Method: Every step in the analytical method must be described and explained. Performance specifications and system suitability tests, defined critical points, and convenient stopping points must be incorporated. Descriptions of equipment and reagents should be written generically, if possible, to avoid dependence on specific brand names and allow the method user to determine suitability of those items in his or her own laboratory. The detailed method written by the Study Director should then be tested by an analyst not previously associated with its development.

Obtaining Participation: Lists of possible participants can be developed through personal contacts, technical societies, trade associations, literature search, and advertisements in the Referee section of AOAC’s magazine, Inside Laboratory Management. Laboratories invited to participate should have personnel experienced in the basic techniques employed; experience with the method itself is not a prerequisite for selection.

Laboratories must realize the importance of the study. A large investment is made in testing the method and this probably will be the only collaborative study of the method that will be performed. Therefore, it is important to have a fair and thorough evaluation of the method.

SUMMARY OF ADOPTION PROCESS

(1) A method is adopted as a First Action AOAC® Official Methods^sm by a Methods Committee after successful completion of an interlaboratory collaborative study, conducted by a Study Director according to AOAC specifications, and after review and recommendation by the General Referee, Statistical and Safety Advisors, Methods Committee, and 2 Official Methods Board members.

(2) A method is adopted as a Final Action AOAC® Official Methods^sm after publication of the method and collaborative study report has allowed further use and testing by the scientific community; review and recommendation by the General Referee and Methods Committee; and a vote by the Official Methods Board.

(3) First and Final Action AOAC® Official Methods^sm may also be revised or repealed.

(4) Notices of all proposed actions and completed actions for AOAC® Official Methods^sm are published in the Referee section of AOAC’s magazine, Inside Laboratory Management, and on the AOAC Web site. Collaborative study reports for new First Action methods are published in the Association journal, Journal of AOAC INTERNATIONAL. All First and Final Action AOAC® Official Methods^sm are published in the compendium, Official Methods of Analysis of AOAC INTERNATIONAL, which is updated annually.

HOW CAN YOU GET STARTED?

Scientists who are interested in development and validation of analytical methods should contact AOAC INTERNATIONAL for more detailed information and notify AOAC INTERNATIONAL of their wish for a volunteer appointment. Anyone with the knowledge, interest, and experience in the subject matter field may be appointed as an AOAC Study Director.

WHO IS AVAILABLE TO HELP?

Every appointment comes with information about staff contacts, names and addresses of the assigned General Referee, Statistics Advisor, Safety Advisor, Methods Committee members, and other Study Directors working on methods in similar areas. The Association magazine, Inside Laboratory Management, is available as a medium to recruit collaborators.

WHERE TO WRITE OR CALL

AOAC INTERNATIONAL
481 N. Frederick Ave, Suite 500
Gaithersburg, MD 20877-2417, USA
Telephone: +1-301-924-7077
Fax: +1-301-924-7089
Internet e-mail: aoac.aoac.org

METHODS PROGRAM STEPS

Study Design: Study Director

Protocol Review: General Referee, Statistics and Safety Advisors, Methods Committee representatives

Collaborative Study: Study Director and collaborators

Study Report: Study Director

Report Review: General Referee, Statistics Advisor, Methods Committee, and 2 Official Methods Board Members

Method Adoption: Methods Committee

Method Publication: Official Methods of Analysis of AOAC INTERNATIONAL

Study Publication: Journal of AOAC INTERNATIONAL

COLLABORATIVE STUDY PROCESS

Method Development—In-House: (Method Choice; Method Optimization; Ruggedness Testing)

Protocol Design: Method Write-Up; Choice of Laboratories; Test Materials; Statistical Design

Study Preparation: Participants; Instructions; Preparation and Shipping of Test Samples

Collaborative Study Execution: Collaborative Analyses; Data and Report Submission

Study Analysis: Data Audit; Outliers; Accuracy; Precision; Conclusions

Final Report: Background; Study; Method; Results; Recommendations

COMMITTEE STRUCTURE

Official Methods Board

(A) Methods Committee on Pesticide and Disinfectant Formulations: General Referees (CIPAC Studies; Disinfectant Formulations; Fungicides and Rodenticides; Herbicides; Insecticides, Synergists, and Repellents); Study Directors

(B) Methods Committee on Drugs and Related Topics: General Referees (Drugs; Drug Residues in Diagnostics and Test Kits; Drug Residues in Foods; Cosmetics; Forensic Sciences); Study Directors

(C) Methods Committee on Additives, Beverages, and Food Process Related Analytes: General Referees (Beverage Alcohol; Food Additives; Flavors; Spices and Other Condiments; Color Additives; Filth and Extraneous Materials in Foods and Drugs); Study Directors

(D) Methods Committee on Natural Toxins and Allergens: General Referees (Mycotoxins; Food Allergens; Marine and Freshwater Toxins); Study Directors

(E) Methods Committee on Food Nutrition: General Referees (Dietary Fiber; Fats and Oils; Infant Formula and Medical Diets; Minerals; Sugars and Sugar Products; Fat-Soluble Vitamins; Water Soluble Vitamins; Nonvitamin Micro-Nutrients); Study Directors

(F) Methods Committee on Commodity Foods and Commodity Products: General Referees (Cereals and Cereal Products; Chocolate and Cacao Products; Dairy Chemistry; Fruits and Fruit Products; Meat and Meat Products; Seafoods; Processed Vegetable Products); Study Directors

(G) Methods Committee on Residues and Related Topics: General Referees (Metals and Other Elements; Multiclass Multiresidue Methods for Organic Compounds; Single Class Multiresidue for Organic Compounds; Radioactivity; Pesticides and Other Chemical Contaminants); Study Directors

(H) Methods Committee on Microbiology: General Referees (Drug- and Device-Related Microbiology; Food Microbiology—Dairy; Food Microbiology—Nondairy; Genetically Modified Organisms; Microbiological Efficacy Testing of Disinfectants; Bacillus anthracis); Study Directors

(I) Methods Committee on Feeds, Fertilizers, and Related Agricultural Materials: General Referees (Antibiotics in Feeds; Drugs in Feeds; Feeds; Fertilizers & Agricultural Liming Materials; Nutrients in Soils; Veterinary Analytical Toxicology; Tobacco); Study Directors

(J) Methods Committee on Environmental Quality: General Referees (Inorganic Methods; Organic Methods; Environmental Microbiology; Environmental Chemistry; Bioassay Methods); Study Directors

(K) Methods Committee on Dietary Supplement: General Referees (Botanicals; Plant Toxins); Study Directors

AOAC COLLABORATIVE STUDY

The following is a summary of the information that is presented in detail in the internationally harmonized document, “Guidelines for Collaborative Study Procedure to Validate Characteristics of a Method of Analysis,” and is given in Appendix D. The document is the basis for an AOAC validation study.

Design of the Collaborative Study

General Principles: The design should attempt to identify and to include the possible sources of significant variability that may occur in actual practice, including between days, between runs, and between calibration curves, if these are significant factors. The best measure of within-laboratory variability is obtained by using blind replicates and/or split levels (Youden pairs). The design must take into account how the data will be analyzed statistically.

Laboratories: Minimum number of laboratories for quantitative analysis.—A minimum of 8 laboratories submitting valid data is needed for a quantitative method (only in special cases involving very expensive equipment or specialized laboratories may the study be conducted with a minimum of 5 laboratories, with the resulting expansion in the confidence interval for the statistical estimates of the method characteristic). Minimum number of laboratories for qualitative analysis.—A minimum of 15 laboratories is needed for qualitative studies reporting on 2 analyte levels per matrix, 5 test samples per level, and 5 negative controls per matrix. It is prudent to include more than the minimum to avoid jeopardizing a study in which results of some laboratories must be discarded.

Test Materials: Minimum number of materials is 5 for quantitative analysis (only when a single level specification is involved for a single matrix may this minimum be reduced to 3). Test materials must be homogeneous (this is critical) and coded at random so that there is no preselection from order of presentation. Analyte levels should be chosen to cover concentration range of interest, especially tolerance limits, specification levels, and likely levels of occurrence.

Materials should be representative of commodities usually analyzed, while being stable and able to withstand the rigors of commercial transportation. Practice test samples should be provided, and reserve test samples should be prepared and preserved to replace lost or damaged items and to permit re-analysis in the case of outliers or abnormal results.

Replication: For within-laboratory variability, independent replication can be ensured by applying one of the following procedures: (1) Split levels (Youden pairs). A pair of materials of slightly different concentration obtained either naturally or by diluting (or by fortifying) one portion of the material with a small amount of diluent (or of analyte). (2) Split levels for some materials and blind duplicates for other materials in the same study. (3) Blind duplicate test samples—randomly coded. (4) Independent materials. Although use of known replicates is a common practice, it is preferable to use the same resources for blind replicates or split levels.

Blanks: When the absence of a component is as important as its presence, when determinations must be corrected for the amount of the component or the presence of background in the matrix, or when recovery data are required, provision must be made for the inclusion of blank materials containing “none” (not detected) of the analyte. It is also important to know the variability of the blank and the tendency of the method to produce false positives.

Analysis and Report

AOAC INTERNATIONAL requires the calculation and reporting of percent recovery (% Rec.), HorRat, repeatability (within-laboratory, sr) and reproducibility (interlaboratory, sR) standard deviations, and repeatability and reproducibility relative standard deviations (RSDr and RSDR, respectively). Specific guidelines and tools are available to aid the Study Director in performing the statistical analysis of the collaborative study data. These include spreadsheet forms for the calculation of performance parameters and a software package for computer calculations from the data.

The final report should contain the purpose of the study and the principles of the method, a brief summary of related work, a description of the collaborative study design, the complete method, and the results and conclusions. The report must also include the names of the study participants and their organizations.