ERP Sulfite Methods-Meeting Materials_4-2020

TABLE OF CONTENTS

ACCESS TO METHOD REVIEW FORM: https://form.jotform.com/52106307924147

I. PRELIMINARY ITEMS

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a. Approved ERP Meeting Roster b. ERP Meeting Draft Agenda c. ERP Meeting Governance Information

d. AOAC Method Manuscript and Format Information

II. PROPOSED METHOD FOR FIRST ACTION OFFICIAL METHODS STATUS a. Total Sulfites in Shrimp – Submitted by Biolan Microbiosensores

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III. SUPPORTING VALIDATION DATA AND TESTING PROTOCOLS a. Supporting Validation Manuscript – Original Approved AOAC Performance Tested Methods SM (PTM) – License Number 031802 b. Supporting Validation Manuscript – PTM Method Extension – Range Extension c. Approved PTM Internal Validation Testing Protocol d. Approved PTM Independent Laboratory Validation Testing Protocol e. AOAC 990.28 – Sulfites in Foods Used for Method Comparison

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IV. APPROVED COLLABORATIVE STUDY PROTOCOL INFORMATION a. Approved AOAC Collaborative Study Protocol b. Stability Study of Test Portions for Collaborative Study Protocol

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c. Data Reporting Forms for Collaborators d. Response to Comments on Protocol

V. BIOLAN INSTRUCTION MANUALS a. BioFISH Instruction Manual

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b. BioFISH Sulfite Instruction Manual with Range Extension

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AOAC ERP for Sulfites in Seafood Methods Roster as of April 2020

OLIVER OU (MEMBER) US Food and Drug Administration (FDA) Oliver.Ou@fda.hhs.gov

CHERYL LASSITTER (CHAIR) US National Oceanic Atmospheric Administration (NOAA) cheryl.lassitter@noaa.gov SNEH BHANDARI (MEMBER) SDB Associates Sdbhandari1@gmail.com KATHERINE CARLOS (MEMBER) US Food and Drug Administration (FDA) Katherine.Carlos@fda.hhs.gov GEORGE JOSEPH (MEMBER) AsureQuality NZ George.Joseph@asurequality.com KATERINA MASTOVSKA (MEMBER) Eurofins Food Integrity & Innovation katerinamastovska@eurofinsus.com

TOM PHILLIPS (MEMBER) Maryland Department of Agriculture tom.phillips@maryland.gov EDMOND SANGANYADO (MEMBER) Shantou University esang001@ucr.edu SPENCER WALSE (MEMBER) US Department of Agriculture (USDA) spencer.walse@ars.usda.gov

DEBORAH McKENZIE (STAFF LIAISON) Sr. Director, Standards & Official Methods SM

AOAC INTERNATIONAL dmckenzie@aoac.org

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EXPERT REVIEW PANEL FOR SULFITE METHODS

Friday, April 24, 2020 Meeting Time: 1 0 :00AM – 12:30PM (Eastern US) ERP Chair: Cheryl Lassitter (US NOAA)

I.

WELCOME AND INTRODUCTIONS (Lassitter) Cheryl Lassiter (US NOAA) will welcome attendees and entertain introductions and establish ERP quorum via roll call by McKenzie (AOAC INTERNATIONAL) . ERP MEETING PROCEDURES OVERVIEW (McKenzie) McKenzie will provide an overview of procedures for the ERP meeting and method review. III. REVIEW OF METHOD AND VALIDATION DATA* The BIOFISH 300 SUL has undergone validation through the AOAC Performance Tested Methods program and has been certified. The manuscript for this method is submitted for consideration by the ERP as Total Sulfites in Shrimp for Official Methods First Action status. a. Presentation on the method and the validation studies to date. b. Lassitter and McKenzie to lead the ERP through a review and discussion of the method for Consideration IV. NEXT STEPS Depending on the consensus and outcomes of the ERP discussion, McKenzie will advise on options for next steps and the ERP will reach consensus on the actions to be taken. V. ADJOURNMENT (Lassitter) II.

Draft meeting agenda is subject to change w/out notice *Items may require a consensus vote of ERP.

Version 1

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REQUIREMENTS FOR ERP SERVICE  Must have demonstrated expertise in the method, technology, analyte/matrix, etc… Be a subject matter expert.  Must be able to attend ERP meetings  Must be able to complete assigned reviews on time  Must be prepared to speak on the method and share reviews during the meeting

 Must be proactive in tracking assigned First Action Official Methods  Must be able to assist in peer reviewing paper for publication  Must sign and submit AOAC Volunteer Acceptance Form  Eligible to serve as a Volunteer Expert in the PTM program

ETHICAL EXPECTATIONS FOR ERP MEMBERS • Respect for your peer ERP members and chair

– Each member has been vetted for expertise relevant to the review of the method(s) in the ERP • Be considerate of each other’s perspectives and points of view • Be considerate of the ERP’s consensus even if you disagree – Inform staff as early as possible if you cannot attend the scheduled ERP meeting • Be considerate in that your absence can impact the quorum of the ERP and its ability to have an official meeting to make decisions – Notify staff and/or disclose in the ERP meeting if you have a direct or perceived conflict of interest for a specific method • Please review AOAC’s policy on Volunteer Conflict of Interest • Respect for Method Authors and Intellectual Property – Each Method Author is encouraged to attend the ERP meeting – Each candidate methods (not yet adopted or published as Official Methods of Analysis of AOAC INTERNATIONAL ) are still the intellectual property of the method author. Therefore, the information is shared only with the vetted ERP members and is available during the meetings. Please do not distribute the information without expressed written permission from an appropriate AOAC staff liaison. – Be clear about and justify how additional recommended work is a requirement for First Action, a requirement for Final Action consideration, or something recommended, but not necessary. – Keep your focus on the science

CANDIDATE METHOD REVIEWS  In your judgment, does the method sufficiently meet the Standard Method Performance Requirements (SMPR) or community ‐ based guidance?

 In your judgment, is the method scientifically sound and can be followed?  In your judgment, what are the strengths and weaknesses of the method?

 In your judgment, how do the weaknesses weigh in your recommendation for the method?  In your judgment, will the method serve well the stakeholder community that will use the method?  In your judgment, is the method/manuscript in AOAC format?  In your judgment, what additional information may be needed to further support the method meeting the SMPR or community ‐ based guidance?  Members of both Committee on Safety and Committee on Statistics serve as advisory resources for all ERPs

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ERP MEETING QUORUM AND CONSENSUS VOTING MEETING QUORUM ERP Meeting Quorum is seven (7) vetted members of the ERP OR 2/3 of the total vetted ERP, WHICHEVER IS GREATER. NO QUORUM, NO MEETING.

POTENTIAL MOTIONS BY ERP FOR CONSENSUS ON METHODS Consensus Decision Motion Adopting a Method* (as is)

To move the candidate method to Official Methods SM First Action status (ERP can include limits to applicability, if substantiated) To move the candidate method for (modified applicability) to Official Methods SM First Action status To delay decision on the candidate method pending reconsideration and resubmission by the method author To approve the following recommendations and to reconsider the method for Official Methods SM First Action status:  Requirements  Suggestions To send the candidate method back to the method author with information that the method requires further work and cannot be considered at this time. To approve the list of requirements for the ERP’s consideration of the method for Final Action

Adopting a method* (modified applicability) Not Ready to Adopt (but very promising) Recommendation as a follow up to the previous motion Not Reay to Adopt (much work is needed) Requirements for ERP Recommendation for Final Action

Each Motion will require a second and a vote by the ERP. * Method must be adopted by unanimous decision of ERP on first ballot. If not unanimous, negative votes must delineate scientific reasons. Negative voter(s) can be overridden by 2/3 of non ‐ negative voting ERP members after due consideration. Abstentions do not count towards vote; in case of multiple abstentions the results will need to be evaluated. Staff will monitor and record consensus voting.

OTHER CAVEATS AND INFORMATION:

Circumstance

Resolution

Method authors and those affiliated with the method under review may be eligible to serve on the ERP

He/She/They must reveal their direct or perceived connection and/or conflict of interest to AOAC staff and the ERP. It is acceptable to recuse oneself and abstain from voting. ERP members of the same affiliation have only one (1) vote counted. Serves as a resource. Members of the Committees on Safety and Statistics also serve as a resource to both the ERP and method authors. Method authors will be invited to recommend three (3) ERP members to conduct peer review of the manuscript.

More than one ERP member may have the same affiliation

Official Methods Board will assign a member to be a liaison to the ERP ERP members may participate in the peer review process of the manuscript publication

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I. General Guidelines Below is the general format of manuscripts with Official Methods or candidate Official Methods for ERP review and AOAC Publications.

A. Before the Method

o Title of the Manuscript o Author names and Affiliations o Abstract o Introduction o Summary of Validation Study Design or Protocol used. B. Method (See section II for the Formatting of Methods in Manuscripts)

C. After the method o Manuscripts should include the following sections: Results and Discussion, Conclusions, Acknowledgments, and References. II. Format and Style for Official Methods The Method portion of the manuscript should be included after the Introduction. Methods must include the following elements: Title of the Method The title should include the Method number, analyte, matrix, technology used and the official status. AOAC will provide the Official Method number after the paper is received for publication as such, authors should use the following 2020.xx

Example:

AOAC Official Method 2020.xx Analyte in Matrix Technology used First Action 2020 (use the year that the method was approved)

Applicability statements Applicability statements should include the list of matrix(es) along with specific matrix types and range or limits of determination or detection. Applicability statements should address utility and limitations on use of method or other information. [Applicable for determination of ….] Caution Statements List any statements of caution after the applicability section. Review all methods for potential hazards. Authors should automatically incorporate safety statements. Decisions regarding inclusion of caution statements should be practical; overuse will be self ‐ defeating. Methods that create toxic, obnoxious or environmentally hazardous fumes and wastes should contain practical directions for disposal. Specify precautions and possible hazards in carrying out method, including safety information on equipment, techniques and practices, and safe handling of chemicals, acids, alkalies, microorganisms, and solvents. Example: Caution: Refer to the material safety data sheets for all chemicals prior to use. Use all appropriate personal protective equipment and follow good laboratory practices. Performance Parameters Include statistical data if the study provides sufficient information with regard to the reliability of the method. All headings should be set in Alphabetical Order i.e., A. Principle, B. Apparatus, C. Reagents. Methods should be laid out in the following order: A. Principle

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Explains scientific premise on which the method is operates specifically the mechanism of the analysis. Explain the purpose of various steps or the basis of unfamiliar or unusual reactions. Include the scope and sensitivity of the method—its applicability to certain types of samples and its non ‐ applicability, because of interference, solubility, or other reasons, to other types of samples. Methods are divided into several descriptive sections i.e., Apparatus, Reagents, Reagent Preparation, Standard Preparation, Sample Preparation, Extraction . The sections should include specifications for necessary laboratory apparatus and reagent preparations. See also Definition of Terms and Explanatory Notes . NOTE: Official Methods will need to include InChi numbers and CAS numbers where appropriate. B. Apparatus Lists the equipment that requires assembly or that has specifications critical to the method performance. Describe equipment in terms of performance characteristics. List the necessary laboratory apparatus. For more information see “ Definition of Terms and Explanatory Notes ” Apparatus should be set as in the following example: (a) Ultra ‐ HPLC (UHPLC) system —Nexera (Shimadzu, Kyoto, Japan) or equivalent LC system consisting of a dual pump system, a sample injector unit, a degasser unit, and a column oven. (b) Triple ‐ quadrupole mass spectrometer —Triple Quad 6500 (Sciex, Framingham, MA) or equivalent tandem MS (MS/MS) instrument. (c) Column —Kinetex C18 core ‐ shell, 2.6 μ m, 2.1 × 50 mm (Phenomenex, Torrance, CA) or equivalent. C. Reagents Do not list common reagents which would ordinarily be expected to be available in a well ‐ equipped analytical laboratory. Reagents without specifications are automatically reagent grade, conforming to the specifications of the American Chemical Society (ACS) when such specifications exist. Reagents should follow the same format as in the above example under Apparatus. D. Reagent Preparation, Standard Preparation, Sample Preparation, Extraction Sections Use these sections for materials requiring directions for preparation, purification, or standardization. Standard compounds will often need specifications or a source of supply. The sections should use the same style and format as in the above example under Apparatus. E. Determination If a method is fairly straight forward or consists only of a single major step, describe all operations under this heading. If the method is complex, however, divide the determinative section into several parts which may be characterized by the type of operation performed. Be sure to identify all critical control points. F. Calculations Include calculations in a method for convenience to avoid the need for looking up factors and deriving equations, particularly when a series of multiple dilutions or aliquots are used at various steps in the method. Take particular care to ensure that there is no ambiguity with regard to the entries in the numerator and the denominator. G. Data Handling A section on data handling should be included if necessary. Example : Report results as μ g/hg to one decimal place or as IU/hg to zero decimal places. Other sections may be included as needed and should be labelled in alphabetical order (i.e., H. Chromatography) Tables and Figures Tables and Figures should be in separate documents and clearly labelled. Do not intersperse tables and figures within the text document. Tables and figures should be numbered consecutively as Table 1, Table 2, Figure 1, Figure 2. For instructions on how to layout Tables and Figures specifications please visit our Instructions to Authors page. Supplemental information Supplemental information is allowed and should be included as a separate Word document clearly labelled. Supplemental information should be cited in the text. Supplemental data may include large tables, figures or appendices. Please note: this information will be available on the online Journal site and will be uploaded as submitted. It will not be copyedited or typeset.

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Detailed Author Instructions for Each Section of Manuscript Including Tables and Figures Please follow these instructions closely; doing so will save time and revision. Note: if the instructions are not followed and the paper is accepted the Editorial Office will return the paper to the author and the instructions will need to be followed before production and publication can begin. If the Section Editor sends notification to author that changes are necessary, it is the author’s responsibility to upload updated files per Section Editor’s comments before approval is granted. 1. Write in clear, grammatical English. 2. Double space all materials. Do not right justify or use proportional spacing; avoid hyphenation. 3. Suggest at least three qualified reviewers (i.e., Expert Review Panel members). Text Please send manuscript text as a Word file. Tables and figures should set as separate files clearly named (i.e., table 1, table 2, figure 1, figure 2 etc.). DO NOT intersperse tables and figures in text. Choose a title that is as descriptive of the paper’s content as possible. Do not try to make the title clever or “catchy.” Use specific terms that will serve as index entries; information services increasingly rely on titles for indexing. Avoid the use in the title of symbols, formulas, and terms that must be defined. Contact AOAC staff in Standards and Official Methods SM or in Publications for style suggestions for method manuscripts if needed. 2. Author information Give the full first name, middle initial, and surname of each author. Do not include academic titles. List the affiliation(s) of the author(s) in a form that will serve as a complete mailing address, including zip code. The corresponding author needs to be identified and e ‐ mail address supplied. Use the affiliation at which the work was performed; if authors have moved to a different affiliation or if any of the authors have moved to a different address, give the address in a footnote, indicated by a superscript Arabic numeral. It is generally assumed that all persons listed as co ‐ authors of a paper or report have made substantial contributions to the work reported. Other supplemental information should be moved to Acknowledgements i.e., supported by a grant or contract, taken from a thesis or dissertation, publication authorized by an institution, or part of the institution’s publication series. 3. Abstract 250 words. Provide specific information, not generalized statements; abstracts must be organized and include the following subheads: Background, Objective, Methods, Results, Conclusions, Highlights. 4. Introduction Include a statement of the purpose of the work, together with enough background to enable the reader to attain the proper understanding and perspective. State the use of the compound being discussed. Cite the work of others which contributed directly to the present paper but do not attempt to survey the entire literature of the field unless the paper is intended to be a review. Consider the following hypothetical questions in deciding what information to include in the introduction: Why was the work done? Why was a method needed? Did any methods exist previously, and if so, why were they not suitable? If a new method had to be developed, upon what established chemical, biological, or physical principles was it based? What general approach was used? Format and Style 1. Title

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If an interlaboratory study is being reported and a background report has already been published, do not repeat all the introductory material; instead, cite the earlier report and go directly to the description of the interlaboratory study. 5. Study Design Include the type of study(ies) that were conducted including, Collaborative Study, Single Laboratory Validation, etc. Include a section sub ‐ heading for each type of study used to develop data in the validation of the method written in the paper with a brief description of the study, and samples used. Do not include the results, just a summary of what was done to evaluate the method and collect the data. Include any notable procedures in the study design. 6. Method Consult section II (Format and Style for Official Methods of Analysis) and section D of this Guide on preparing methods. Follow these general principles: Write the method in imperative style (“add 10 mL”; evaporate the solution to dryness,” instead of “10 mL was added;” “the solution was evaporated to dryness”). Place important reagents and apparatus in separate sections (or combine them into one separate section) before the actual determination; indicate specifications, source of supply, and instructions for preparation of working solutions where pertinent. List, each reagent or piece of apparatus as a separate item. Provide enough detail so that the reader can repeat the method if he wishes, and indicate how the results are to be calculated, if a calculation is required. 7. Results/Discussion Discuss the results fully enough so that their significance is evident, and conclusions can be drawn from them, but do not use the Discussion merely to repeat data that are given in tables and figures. Provide a statistical treatment of the data if an interlaboratory study has been conducted and where appropriate in other reports and papers. Indicate whether certain data have been rejected and name the statistical test which was the basis for rejection. Try to provide an explanation for unexpected results if possible or, if you cannot explain them, state this fact. In general, do not present the same quantitative data in both a table and a figure in the same paper, because this is expensive and a waste of journal space and money; choose one form or the other. (The exception to this rule is Youden’s diagrams.) Although it is difficult to generalize consider using figures where trends or relationships are especially important and using tables where individual results should be reported, e.g., interlaboratory studies. If a method is being studied, give both recovery data obtained on known samples (usually prepared by the author) and results on unknown samples of the type encountered in regulatory work. Except in unusual cases, do not include straight ‐ line graphs; instead, state in the text the curve is linear in the range of interest. Cite tables and figures consecutively in text with Arabic numerals. 8. Acknowledgments Give brief thanks (no social or academic titles). Financial aid should be acknowledged in this section. 9. References Submitted papers or unpublished oral presentations may not be listed as references; cite them in text as unpublished data or personal communications. Cite all references to previously published papers or papers in press in numerical order in text with number in parentheses on ‐ line (not superscript). List references numerically in References in exactly (arrangement, punctuation, capitalization, use of ampersand, etc.) the same styles of examples shown below or see recent issue of the Journal for less often used types of entries. Follow Chemical Abstracts for abbreviations of journal titles.

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10. Tables and Figures Tables

Tables should be supplied in an editable format (e.g., Microsoft Word), not as an image file. Avoid excessive formatting and the use of tabbed spacing to indicate alignment and ensure that any formatting or superscript symbols such as asterisks are explained in the table footnote. Unless the journal’s house style indicates otherwise, provide units in column or row headers, rather than in the table body. Consider the size and layout of the printed journal (or online PDF) when deciding on the dimensions of your table. All tables provided should be cited within the article text. Figures Each figure should be submitted as its own file. Supply figures at the intended print size. Resolution at this size should be no less than 300dpi for halftone images, 600dpi for composite images, and 1200 dpi for line art, and at no less than 1080px width. For typical print page sizes please visit https://academic.oup.com/journals/pages/authors/preparing_your_manuscript . The dimensions of the printed journal or online PDF should also be considered when choosing figure layout, to minimize white space when reproduced at the intended size. Most standard figure formats are acceptable, but .tiff is recommended for halftone and composite images, .eps for line art and vector graphics. Images embedded into Microsoft Word are often not good quality. Images created with multiple layers should be flattened to a single layer prior to submission. Multi ‐ paneled figures should be supplied as a single file with each panel lettered clearly, according to journal style. Avoid placing letters over shaded areas if possible. Miscellaneous Abbreviation for liter is L; abbreviation for micron is μ . Do not italicize common Latin expressions such as et al. and in vitro; for nomenclature of spectrometry, gas chromatography, and liquid chromatography, follow practice of American Society for Testing and Materials. Do not use numbering in your headings or subheadings. Major headings should be set in bold font, secondary

headings in italic font, and third level headings should be set using alphabetical lists (a) . The Journal prefers the use of the front solidus (/) do not use superscript  ‐ 1 (i.e., μ g/kg).

Footnotes Avoid use of footnotes to text.

Special Instructions for Journal Articles containing Official Methods of Analysis First and Final Action Methods First and Final Action Official Methods will need to include InChi numbers and CAS numbers where appropriate. Additional Author Guidelines For additional information on manuscript preparation please visit the Oxford Academic website at: https://academic.oup.com/journals/pages/authors/preparing_your_manuscript

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PROPOSED METHOD FOR FIRST ACTION STATUS

AOAC Official Method XXXX.XX Total Sulfites in Shrimp Enzymatic Electrochemical Biosensor

BIOFISH 300 SUL First Action XXXX

[Applicable to the determination of total sulfites in the range 7 ‐ 300 mg/kg, measured as SO 2 , in raw shrimp with heads (30 ‐ 300 mg/kg range only), raw headless shrimp, and cooked shrimp.] Caution : Sulfites may cause allergy ‐ like reactions including sneezing, runny nose or wheezing; or asthma symptoms in people with underlying asthma; or rarely hives or anaphylaxis. The use of personal protective equipment (PPE) including gloves, laboratory coat and protective eyewear is recommended. See Tables X and X for a summaries of results of the AOAC Performance Tested Methods studies, supporting First Action status acceptance of the method. [ J. AOAC Int . XX , xxx (2019)]. A. Principle The BIOFISH ‐ 300 SUL method is based on an enzymatic electrochemical biosensor for the specific detection of sulfite. The test method consists of the extraction of sulfite in an aqueous based solution and the subsequent quantification by the biosensor after previous calibration. The biosensor is a system that incorporates a biochemical sensing element (the Biotest, based on a specific oxidative enzyme) in intimate contact with or in close proximity of a transducer system that relates the concentration of an analyte to a measurable signal. The addition of the analyte causes sequential redox reactions that involve a release of electrons proportional to the concentration of the analyte. Thus, these biosensors combine the specificity and the selectiveness of enzyme ‐ analyte reactions with highly sensitive electrochemical transduction. B. Test Kit ( a ) BIOFISH 300 SUL.— Comprised of the following individual components available from Biolan Microbiosensores S.L.: ( 1 ) Biotest.— BTFSUL (30 ‐ 300 mg/kg) or BTFSULAL (7 ‐ 30 mg/kg) for 20, 50, 100, or 125 analyses. ( 2 ) SUL Calibration Kit.— KITCFSUL. ( 3 ) SUL Measurement Kit.— KITMFSUL1 (for up to 160 analyses) or KITMFSUL2 (for up to 450 analyses). ( 4 ) SUL Extraction Solution.— SEFSUL1 (for up to 50 analyses) or SEFSUL2 (for up to 100 analyses). C. Apparatus ( a ) BIOFISH 300 Biosensor .—BF300 ‐ 004, includes biosensor, 2 reference electrodes, electrode storage solution, and 2 cuvettes. ( b ) Analytical balance.— Capable of measurement to two decimal places (g).

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( c ) Mincer or food processor.— Mini Chopper FC ‐ 5140 or equivalent. ( d ) Sample homogenizer.— Ultraturrax® or equivalent. ( e ) Adjustable pipets with tips.— Capable of delivering 20 ‐ 200 μ L and 500 ‐ 5000 μ L.

D. Additional Supplies and Reagents ( a ) Plastic tubes. —50 mL. ( b ) Graduated cylinder.– 25 mL.

( c ) Volumetric flasks .—20 mL and 50 mL ( d ) Glass or plastic bottles with caps. —1 ‐ and 2 ‐ L sizes.

E. Precautions ( a ) Proceed to equipment Reconditioning, Activation and Calibration right before beginning the analysis, on a daily basis, and with well ‐ tempered Solutions, Reagents and Biotest. ( b ) In each process, ensure that the three components of the electrochemical cell (Biotest, reference electrode and counter electrode) are well ‐ submerged in the SUL Measurement Solution and that the reference electrode is well ‐ connected. If a breakdown occurs with the reference electrode, or if work is carried out without it being connected, it will be detected that the current intensity inside is very unstable and with variable intensity peaks. If this is the case, stop the measurement and discard both the Biotest and the reference electrode. ( c ) The main screen Stand By option is used when an intentional pause is to be taken in the analysis routine. ( d ) If the result of an analysis is higher than the upper limit of the Measurement Range being worked in (for example, a value of 320 mg/kg in the range of 30 ‐ 150 mg/kg), change the content of the cuvette and put the equipment into Standby mode for at least 30 seconds before moving forward with the following analysis. ( e ) It is important to store the reference electrode in the Storage Solution supplied along with the electrode (4 M KCl) and in a vertical position whenever not in use. Improper storage may lead to deterioration over a short space of time. ( f ) Follow all local, state, and federal regulations for proper disposal. F. Electrode Hydration and Storage ( a ) Biotest . ‐‐ The Biotest electrodes are shipped at room temperature and vacuum packed with silica gel crystals to protect them from atmospheric humidity. Upon receipt, store unopened at 3 ‐ 8°C until expiration date. To hydrate the Biotest prior to using (this process should be carried out at least 24 hours prior to use): ( 1 ) Remove the silica gel crystals from inside the tube. ( 2 ) Fill the tube with SUL Measurement Solution until the base of the Biotest is submerged. ( 3 ) Add 20 μ L of SUL Calibration Reagent. ( 4 ) Once hydrated, the Biotest must be stored vertically at 3 ‐ 8°C for a maximum of 15 days. ( b ) Reference electrode (ELEREFN) . ‐ Store in 4M KCl at room temperature in the dark and in an

upright position until use. G. Reagent Preparation

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( a ) SUL Measurement Solution. ‐‐ Dissolve the contents of 1 tube of MFSUL1 in 250 mL water for up to 20 analyses or dissolve the contents of 1 tube of MFSUL2 in 1 L water for up to 90 analyses. Store at room temperature and use within 15 days. ( b ) SUL Extraction Solution. ‐ ‐ Dissolve the contents of 1 tube of SEFSUL1 in 1 L water for up to 50 analyses or dissolve the contents of 1 tube of SEFSUL2 in 2 L water for up to 100 analyses. Store at room temperature and use within 15 days. ( c ) SUL Calibration Reagent . ‐‐ Dissolve contents of 1 tube of CFSUL with 10 mL SUL Extraction Solution. Store at 3 ‐ 8°C and use within 7 days. ( d ) SUL Standard 1 . ‐‐ For the 30 ‐ 150 mg/kg range, prepare SUL Standard 1 by diluting 53.4 μ L SUL Calibration Reagent to 20 mL with Extraction Solution. Store at room temperature. Prepare fresh daily. ( e ) SUL Standard 2 ‐‐ For the 50 ‐ 300 mg/kg range, prepare SUL Standard 2 by diluting 100 μ L SUL Calibration Reagent to 20 mL with Extraction Solution. Store at room temperature. Prepare fresh daily. ( f ) SUL Standard 3 . ‐‐ For the range 7 ‐ 30 mg/kg, prepare SUL Standard 3 by diluting 33.4 μ L SUL Calibration Reagent to 50 mL with Extraction Solution. Store at room temperature. Prepare fresh daily. H. Sample Preparation ( a ) Preparation of shrimp (raw and boiled). ‐‐ Clean and dismantle the carapace and cephalothorax and other nonedible parts of the exoskeleton and the visible digestive tract (leave the hepatopancreas in case its measurement is required, only for the 30 ‐ 150 and 50 ‐ 300 mg/kg range) from 15 ‐ 20 shrimp. Homogenize the bulk sample with the aid of a mincer. Add 2 g of the homogenized sample to a plastic tube. ( b ) Sample Extraction. ‐ ‐ Add 18 mL of SUL Extraction Solution. Homogenize the contents of the plastic tube with the aid of an ultra ‐ turrax (8000 ‐ 9000 rpm 15 ‐ 20 seconds). Leave to stand for at least 15 minutes at room temperature. Sample extractions are stable up to 1 hour at room temperature. I. Biosensor Setup ( a ) For low range (7 ‐ 30 mg/kg) testing, choose the Biotest BTFSULAL. For medium (30 ‐ 150 mg/kg) and high (50 ‐ 300 mg/kg) range testing, choose the Biotest BTFSUL. Ensure Biotest is prepared as in F . ( b ) Insert the Biotest and reference electrodes into their respective connectors (place the Biotest into the carrier, tightening it with the screw), ensuring that they are well connected. For safety reasons, it is recommended to never disconnect the reference electrode. When the equipment is disassembled after analyses are completed, the reference electrode remains protected with the Storage Solution supplied (4 M KCl). ( c ) Fill the measurement cuvette with 10 mL of SUL Measurement Solution and place it in the cuvette so that all components in the electrochemical cell are submerged. Connect the equipment to a 100 ‐ 240 VAC stable current power outlet and press the power button located next to the screen for 2 seconds. The menu main screen will appear. ( d ) In the BIOTEST option, select the analyte, press », enter the Biotest Code (this code is located on the Biotest identifying label), press » and finally select the measurement range with which you are going to work (7 ‐ 30 mg/kg; 30 ‐ 150 mg/kg; or 50 ‐ 300 mg/kg) and press SAVE. ( e ) From the main screen, the ANALYSIS option leads to the analysis menu: Recondition, Activate, Calibrate, Standard, Verifying Solution, Turrax/Sample, and Stand By. J. Reconditioning, Activation and Calibration of the Biotest

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Once per day, before analyzing samples, the Biotest must be reconditioned, activated, and calibrated. ( a ) Reconditioning. ‐‐ ( 1 ) Press Recondition, verify the proper placement of the cuvette with the 10 mL of SUL Measurement Solution, add sulfite standard (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range) into the measurement cuvette and press CONTINUE. ( 2 ) Wait for the equipment to emit an acoustic signal, press CONTINUE, and once again inject sulfite standard (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range) into the measurement cuvette and wait for the process to end. The message “Biotest reconditioned. Ready to activate” will appear on the screen. ( 3 ) Replace the contents of the cuvette with 10 mL of SUL Measurement Solution. ( b ) Activation. ‐‐ ( 1 ) Press activate, verify the proper placement of the cuvette and press CONTINUE to begin the activation. ( 2 ) Wait for the equipment to emit an acoustic signal, press CONTINUE, and inject sulfite standard (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range) into the measurement cuvette and wait for the process to end. The message “Biotest activated. Ready for calibration” will appear on the screen. ( 3 ) Replace the contents of the cuvette with 10 mL of SUL Measurement Solution. ( c ) Calibration . ‐‐ ( 1 ) Press calibrate, verify the proper placement of the cuvette with 10 mL of SUL Measurement Solution, and press CONTINUE. ( 2 ) Wait for the equipment to emit an acoustic signal. Press CONTINUE and add sulfite standard into the measurement cuvette (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range). This is addition 1. Note : It is important to add the Calibration Standard immediately after pressing the CONTINUE button to record the current intensity correctly at the time of adding it. ( 3 ) Wait for the equipment to register the change in intensity and emit an acoustic signal again. Press CONTINUE and add sulfite standard again (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range). This is addition 2. ( 4 ) Wait until the parameters of the calibration appear on the screen together with a message of “calibration OK” or “calibrate again”. ( 5 ) Replace the contents of the cuvette with 10 mL of new SUL Measurement Solution. If the regression is not valid (“calibrate again” appears), change the measurement cuvette, put it on Stand by for 30 seconds and calibrate again. If the regression is valid (“calibration OK” appears), continue to Standard Measurement. ( d ) Standard measurement. ‐‐ The calibration status should be tested using the STANDARD option after every 10 ‐ 15 measurements or after the device has not been running for 30 minutes. ( 1 ) Press Standard in the analysis option, check that the cuvette is correctly fitted and filled with SUL Measurement Solution and press CONTINUE to begin the Standard measurement. ( 2 ) Wait until the device records the measurement target and it issues a sound signal. Press CONTINUE and add the Calibration Standard (4 mL of SUL Standard 3 for the 7 ‐ 30 mg/kg range; 2 mL of SUL Standard 1 for the 30 ‐ 150 mg/kg range; or 1 mL of SUL Standard 2 for the 50 ‐ 300 mg/kg range) into the measuring cuvette. ( 3 ) Wait until the “Biosensor calibrated” or “Biosensor recalibrate” message appears. (4) Replace the contents of the cuvette with 10 mL of new SUL Measurement Solution. If “Biosensor calibrated” message appeared, proceed to Determination . If “Biosensor recalibrate” message appeared, return to Step J ( c ) Calibration .

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K. Determination ( a ) Press the option ‘Turrax’ (for 30 ‐ 150 or 50 ‐ 300 mg/kg range) or the option ‘Sample’ (for 7 ‐ 30 mg/kg range). Check that the cuvette is correctly fitted and filled with 10 ml of SUL Measurement Solution and press CONTINUE to begin the sample measurement. ( b ) Wait until the device records the measurement target and it issues an acoustic signal. Press CONTINUE and pipette the clear supernatant of the extracted sample (4 mL for the 7 ‐ 30 mg/kg range; 2 mL for the 30 ‐ 150 mg/kg range; or 1 mL for 50 ‐ 300 mg/kg range) into the measuring cuvette. ( c ) Wait until the process ends and the device shows the result of the analysis. ( d ) Replace the contents of the cuvette with 10 mL of new SUL Measurement Solution. L. Interpretation and Test Result Report ( a ) The Biosensor expresses the analysis result in units of mg of sulfite (measured as SO 2 ) per kg of shrimp, taking into account the dilution involved in the extraction of the sample. ( b ) To save the result of an analysis, press SAVE and enter a code. ( c ) To view the saved results, enter the RECORDS option of the main menu screen . Saved results can be downloaded to a computer. ( d ) To delete the saved results, go to RECORDS and press DELETE ALL.

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Biofish-300 SUL: AOAC Performance Tested Method SM 031802

Abstract The Biofish-300 SUL method is a highly specific enzymatic biosensor for the rapid quantification of sulfite, measured as SO 2 content, in crustacean. Sulfite in raw shrimp head on, head off and boiled shrimp was analyzed and performance was examined using naturally contaminated and spiked samples, by comparisons with OMA 990.28 methods. Linearity, selectivity, matrix, consistency, and robustness were evaluated. All results were within acceptable ranges except robustness, which reflected deviation in sample volume and ultraturrax time compared to the standard assay procedures. Accuracy, assessed as a comparison of the Biofish results to the OMA results, ranged from 82 to 115% in all samples except for fortified raw shrimp head on where the low level yielded an accuracy of 138 %. The method bias was in general negative in both incurred and fortified high levels, and slightly positive in incurred low levels. Repeatability was very good as shown by the low RDSr values demonstrating acceptable repeatability precision with results less than 10 % in most of the evaluated values. Regression analyses showed a good correlation between the Biofish and OMA methods with R 2 > 0.99 in all cases.

Method Authors Sandra Salleres, Irune González, Naiara Linaza, Erlantz Ramos, Mónica Fernández, Roberto González, Sonia Maza, and Arrate Jaureguibeitia

Submitting Company Biolan Microbiosensores S.L. Parque Tecnológico de Bizkaia, Laida kalea, 409. 48170 Zamudio, Vizcaya. Spain. biolanmb@biolanmb.com (+34) 946 57 41 61

Independent Laboratory Eurofins Nutritional Analysis Center- Des Moines, IA 2220 Rittenhouse St., Des Moines, IA 50321, USA Phone: 515-265-1461 Contract Laboratory Anfaco Cecopesca Crta. Colexio Universitario, 16, 36310 Vigo - Pontevedra contacto@anfaco.es (+34)986469301

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Reviewer(s) Jeffrey Van de Riet, Canadian Food Inspection Agency Cheryl Lassitter, National Oceanic and Atmospheric Administration, US Department of Commerce Sneh Bhandari, Merieux NutriSciences, Crete, IL

Scope of method Target analyte .-Sulfite, measured as SO 2 content. Matrixes .-Raw shrimp head on, raw shrimp head off and boiled shrimp.

Claims .-Quantification of sulfite from 30 to 150 mg/kg and 50 to 300 mg/kg. Within these ranges, the Biofish method was equivalent to OMA 990.28, the optimized Monier-Williams method for determination of sulfites in foods (4). The method is not intended for regulatory use.

Intended users -

Food laboratories.

- Technical staff of fish and aquaculture companies, including primary and processing sector.

Definitions

a) Linearity Defines the ability of the method to obtain test results proportional to the concentration. Linearity of the method extending beyond the set of standards or calibrators supplied with the kit. b) Selectivity Ability of the method to detect analyte without interference from matrix or other components of similar behavior. c) Recovery Recovery is the ratio of the mean method result to the true value, expressed as a percentage, [mean cand /known spike] x 100 or [mean ref /known spike] x 100. d) Accuracy Accuracy is the ratio of the mean candidate method result to the reference method result, expressed as a percentage, [mean cand / mean ref ] x 100. e) Bias Bias is the difference between the candidate method mean result and the true value, mean cand – known spike. f) Relative Bias Bias is the difference between the candidate method mean result and the mean reference method result, mean cand – mean ref . g) Standard Deviation of Repeatability s r = n

2

1 X X n − − i

∑

(

)

i

1

=

where X i is the i th value, Xbar is the mean value, and n is the number of values.

2

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h) Relative Standard Deviation of Repeatability

RSD r = [s r /mean cand ] x 100

i) Limit of detection: LOD LOD = X +

( ) b s m

0 3.3 1 1.65 −

0 X

b s

where is the mean analytical value of the known negative matrix;

is the intercept of the

m

plot; and is the slope of the plot.

j) Limit of quantitation: LOQ LOQ = 3 x LOD

Principle of the Method

The BIOFISH-300 SUL is an enzymatic biosensor for the specific detection of sulfite. The test method consists of the extraction of sulfite in measurement solution and subsequent quantification by the biosensor after previous calibration. The enzymatic biosensor is a system that incorporates a biochemical sensing element (based on a specific oxidative enzyme) in intimate contact with or in close proximity of a transducer system that relates the concentration of an analyte to a measurable signal. The addition of the analyte causes sequential redox reactions that involve a release of electrons proportional to the concentration of the analyte. Thus, these biosensors combine the specificity and the selectiveness of enzyme-analyte reactions with highly sensitive electrochemical transduction.

General Information Sulfites are some of the oldest and most widespread preservatives in our food supply. They were used in Greek and Roman times in wine, but it was only in the 1880s that their use in as preservatives in meats was pioneered by Australian and South American beef producers wanting to ship their products to England. The use of sulfites in fruit and vegetables became common with the growth of the processed food industry in the twentieth century.

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Sulfites are compounds that contain the sulfite ion SO 3 2- . Sulfites and sulfiting agents are widely used as additives in the food and pharmaceutical industries, thanks to their preservative, antioxidant and antimicrobial activity. COMMISSION REGULATION (EU) No 1129/2011 of 11 November 2011 amending Annex II to Regulation (EC) No 1333/2008 established a list of food additives and their limits in various matrixes including sulfites in seafood:

Unprocessed molluscs and crustaceans

E- number

Name

Maximum level (mg/l or mg/kg as appropriate)

Footnotes

Restrictions/exceptions

only fresh, frozen and deep-frozen crustaceans and cephalopods; crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family up to 80 units only crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family between 80 and 120 units

E 220-228 Sulphur dioxide — sulfites

150

(3) (10)

E 220-228 Sulphur dioxide — sulfites

200

(3) (10)

E 220-228 Sulphur dioxide — sulfites only crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family over 120 units (3) Maximum levels are expressed as SO 2 relate to the total quantity, available from all sources, an SO 2 content of not more than 10 mg/kg or 10 mg/l is not considered to be present. (10): Maximum limits in edible parts. 300 (3) (10)

Processed fish and fishery products including molluscs and crustaceans

E- number

Name

Maximum level (mg/l or mg/kg as appropriate)

Footnotes

Restrictions/exceptions

only Boiled crustaceans and cephalopods only Boiled crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family up to 80 units only Boiled crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family between 80 and 120 units only dried salted fish of the “Gadidae” species

E 220-228 Sulphur dioxide — sulfites

50

(3) (10)

E 220-228 Sulphur dioxide — sulfites

135

(3) (10)

E 220-228 Sulphur dioxide — sulfites

180

(3) (10)

E 220-228 Sulphur dioxide — sulfites

200

(3) (10)

E 220-228 Sulphur dioxide — sulfites only Boiled crustaceans of the Penaeidae, Solenoceridae and Aristaeidae family over 120 units (3) Maximum levels are expressed as SO 2 relate to the total quantity, available from all sources, an SO 2 content of not more than 10 mg/kg or 10 mg/l is not considered to be present. (10): Maximum limits in edible parts. 270 (3) (10)

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Materials and Methods

Test Kit Information

a) Kit Name .—Biofish-300 SUL b) Catalog Number .— BF004 c) Ordering Information i. Worldwide. –

Biolan Microbiosensores S.L. Parque Tecnológico de Vizcaya, Laida kalea, 409. 48170 Zamudio, Vizcaya. Spain

(+34) 946 57 41 61 www.biolanmb.com biolanmb@biolanmb.com

d) Test Kit Components i. BTFSUL20: SUL Biotest for 20 analyses + SUL1 Extraction Reagent (SEFSUL1) for 20 analyses. ii. BTFSUL50: SUL Biotest for 50 analyses + SUL1 Extraction Reagent (SEFSUL1) for 50 analyses. iii. BTFSUL100: SUL Biotest for 100 analyses + SUL2 Extraction Reagent (SEFSUL2) for 100 analyses. iv. BTFSUL125: SUL Biotest for 125 analyses + SUL2 Extraction Reagent (SEFSUL2) for 125 analyses. v. KITCFSUL: SUL Calibration Kit (4 tubes with Calibration Reagent (CFSUL)). vi. KITMFSUL1: SUL1 Measurement Kit (8 tubes of Measurement Reagent (MFSUL1) for a total of 160 analyses). vii. KITMFSUL2: SUL2 Measurement Kit (5 tubes of Measurement Reagent (MFSUL2) for a total of 450 analyses).

viii. ELEREFN: Reference electrode and storage solution (SOLALMER). ix. KITVFSUL: SUL Verifying Kit (4 tubes of Verifying Reagent (VFSUL).

e) Additional supplies and reagents i. Distilled water

ii. Plastic tube .-Falcon, 50 mL iii. Plastic Test tube.- 25 mL

Apparatus

a) Biofish Biosensor .-BF300-004: Biofish 300 sulfite. b) Analytical balance. -Capable of measurement with two decimal places (g). c) Mincer.- d) Sample homogenizer .-Ultraturrax IKA T-25, or equivalent. e) Adjustable pipettes .-Capable of delivering 20-200 μL and 500-5000 μL.

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