OMA Protocol Review: OMAMAN-56 Sulfites in Shrimp

Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

AOAC International Official Methods of Analysis SM Program

Determination of Sulfites in Shrimp by the BIOFISH 300 SUL Method: Collaborative Study Protocol

February 2019 Version 6

Prepared by: Sharon Brunelle AOAC Contractor

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Table of Contents

1. Introduction 1.1. Description of the BIOFISH 300 SUL 1.2. Summary of PTM Study (PTM 031802) 2. Collaborative Study 2.1. Study Director for the Collaborative Study 2.2. Collaborators

2.3. Collaborative Study Design 2.4. Test Portion Preparation

2.5. Test Portion Analysis 2.6. Reporting Raw Data 2.7. Analyzing Raw Data 2.8. Report

3. Appendices

3.1. Instructions to Collaborators 3.2. Study Materials and Apparatus 3.3. BIOFISH 300 SUL Method in OMA Style 3.4. Collaborator Comment Form 3.5. Data Report Form 3.6. OMA 990.28 Reference Method

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1.0 Introduction Sulfites are widely used preservatives in foods. Concentrations at or above 30 mg/kg are needed in order to control melanosis in shrimp. Current US regulations require labeling of seafood products when sulfite concentrations are in the range 10-100 mg/kg. Sulfite is disallowed at concentrations higher than 100 mg/kg and does not have to be declared at concentrations below 10 mg/kg. In the EU, maximum allowable limits for sulfites in crustaceans range from 50 mg/kg to 300 mg/kg, depending on the family of crustacean and the type of processing, if any. Sulfites are a group of chemicals and levels are expressed as SO 2 equivalents. The current US regulatory method is the optimized Monier-Williams method (OMA 990.28), which requires refluxing with HCl to convert sulfites to SO 2 , followed by oxidation of SO 2 to H 2 SO 4 , which is then determined by titration. The method has low throughput and is tedious to conduct. The BIOFISH 300 SUL method was PTM certified for an overall range of 30-300 mg/kg (comprised of two ranges, 30-150 mg/kg and 50-300 mg/kg), which does not allow it to be used for regulatory purposes but does allow it to be used by seafood producers to ensure that sulfite levels are below the maximum allowed in the US or EU. A range extension single lab validation (SLV) study was recently performed. The goal of the range extension SLV was to validate a lower quantitative range (7-30 mg/kg) in order to extend the method down to the lower regulatory level (10 mg/kg) that triggers the requirement for labeling. The original PTM report and the range extension report are provided for your review along with this collaborative study protocol. The goal of the collaborative study is to estimate the reproducibility of the BIOFISH 300 SUL method over the full range (7–300 mg/kg). Performance will be measured by repeatability and reproducibility precision of the method across at least 8 collaborators. The claimed matrices include raw shrimp with head, raw headless shrimp, and boiled shrimp. 1.1 Description of the BIOFISH 300 SUL Method The BIOFISH 300 SUL is an enzymatic biosensor intended for the determination of sulfite in raw shrimp with head, raw headless shrimp, and boiled shrimp, measured as SO 2 . The technology is based on the development of Biotest, a working electrode that immobilizes analyte-specific enzyme(s) together with other electroactive components to produce a change in the electrical current when these components come into contact with the analyte. The BIOFISH300 instrument is the platform for use with the Biotest working electrode, the reference electrode, a measurement cuvette, and extraction, calibration and measurement reagents. Following activation of the Biotest electrode, the electrode is calibrated, and then analyses on unknown samples are carried out. For each measurement, a baseline is determined and subtracted from the reading with calibrator or sample added. Sample preparation involves extraction of a 2 g homogenized sample with 18 mL of Extraction Solution with the aid of an UltraTurrax. A 1-, 2-, or 4-mL aliquot of extract, depending on the target concentration range, is added to the cuvette containing 10 mL of SUL Measurement Solution for sulfite determination. 1.2 Summary of PTM Validation

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The BIOFISH 300 SUL was evaluated by the AOAC Research Institute and granted PTM certification #031802. The performance of the method in 3 matrices was compared to that of OMA 990.28, Sulfites in Foods – Optimized Monier-Williams Method. The validated matrixes included raw shrimp with heads, raw headless shrimp, and cooked shrimp. The method developer studies included linearity, LOD, LOQ, accuracy, bias, recovery, precision, selectivity, lot–to-lot consistency, stability, and robustness. The matrix studies included both incurred and fortified (metabisulfite) matrices. Due to the low throughput and OMA status of the reference method, OMA 990.28 was carried out on 3 replicate test portions per concentration, while the BIOFISH 300 SUL method was carried out on 5 replicate test portions per concentration and was performed on two different days by two different analysts. Anfaco Cecopesca conducted the official analysis with the OMA method for the method developer. The independent laboratory study conducted by Eurofins Nutrition Analysis Center used raw headless shrimp fortified with metabisulfite, with 3 replicates per concentration tested by the OMA method and 5 replicates per concentration tested by the BIOFISH 300 SUL method. Performance indicators included bias, accuracy, recovery, repeatability precision, LOD and LOQ. Accuracy was assessed as (mean Biofish /mean OMA )*100 and recovery was assessed as (mean Biofish /fortification)*100. Accuracy ranged from 82 to 115% in all samples except for fortified raw shrimp with head 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 cases. Regression analyses showed a good correlation between the BIOFISH and OMA methods with R 2 > 0.99 in all cases. The original PTM report and the range extension report are provided for your review along with this collaborative study protocol. 2.0 Collaborative Study The Collaborative Study will follow OMA Appendix D guidelines. 2.1 Study Director The Study Director is: Sandra Salleres Biolan Microbiosensores Tel.: +34 946 574 161 ssalleres@biolanmb.com Collaborators A minimum of 8 valid data sets are needed for the successful completion of this study, so 10-12 collaborators will be solicited to participate. All collaborators have been, or will be, trained on the BIOFISH 300 SUL method. There may be up to two collaborators per laboratory working 2.2

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independently and all laboratories will preferably be in the EU for ease of shipping samples and equipment. An effort will be made to recruit both government and private laboratories. In addition, 3 collaborators will be recruited for the OMA method, but these may be collaborators that are not performing the Biofish method. A sample letter to collaborators can be found in Appendix 3.1. This letter will be sent to each collaborator prior to the start of the study. In addition, a conference call will be held with collaborators to review the study protocol in detail and answer any questions before the start of the study. 2.3 Collaborative Study Design 2.3.1 The study will include 6-12 test sites, with no more than 2 collaborators at any one site. The 2 collaborators may be 2 analysts employed at that site but working independently. 2.3.2 Six materials will be analyzed in the collaborative study. A material is an analyte/matrix/concentration combination. The materials will all be incurred samples sourced from seafood producers. Two materials will be used to test each method range. The low range, 7-30 mg/kg, is validated only for boiled shrimp and raw shrimp with heads off. Boiled shrimp tend to have the lowest sulfite levels and shrimp with heads on tend to have the highest sulfite levels due to the fat content in the heads. The proposed Study Materials in Table 1 take these facts into account. 2.3.3 Collaborators will each receive 2 replicate test portions of each material for a total of 12 test portions as described in Table 1. All test portions for each collaborator will be randomized and blind coded. The randomization will be different for each collaborator. A test portion inventory sheet will be shipped with each test portion set to verify receipt of correct test portions and to indicate which blind coded test portions to use for which method range. 2.3.4 In addition, three collaborators will analyze 2 replicates of each material by the OMA 990.28 reference method. Table 1. Test portion set for each collaborator Material* Test Portion Size Number of Test Portions Method Range No. Matrix Sulfite Concentration 1 Boiled shrimp 7-12 mg/kg 2 g 2 7-30 mg/kg 2 100-150 mg/kg 2 g 2 50-300 mg/kg 3 Raw shrimp with heads off 15-20 mg/kg 2 g 2 7-30 mg/kg 4 40-50 mg/kg 2 g 2 30-150 mg/kg 5 Raw shrimp with heads on 80-100 mg/kg 2 g 2 30-150 mg/kg 6 250-300 mg/kg 2 g 2 50-300 mg/kg *All materials are incurred matrix.

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2.4 Study schedule 2.4.1 Instruments and test kits will be provided to collaborators in the weeks before the study begins. Training will be provided at a date and time convenient for both parties. Fortified or incurred shrimp samples different from the collaborative study materials can and should be prepared and used as training samples to demonstrate proficiency. 2.4.2 Study test portions will be shipped on a Monday by overnight courier to each test site for arrival on a Tuesday. The specific dates will be determined once collaborators are confirmed. 2.4.3 All collaborators will begin analyses on that Wednesday. 2.4.4 Data forms will be completed and emailed to the Study Director within 1 week of initiation of analyses. Table 2. Study Schedule (Specific Dates TBD)

Receipt of BIOFISH 300 SUL

Submission of Data Report Form Week 2, Wednesday

Collaborator Activity

Receipt of Test Portions

Initiation of Analyses Week 1, Wednesday

Training

Week 1, Tuesday

Date

TBD

TBD

2.5

Test Portion Preparation

2.5.1 Obtain a sufficient quantity of incurred raw shrimp with heads on, raw shrimp with heads off, and boiled shrimp at the approximate sulfite concentrations in Table 1. 2.5.2 Homogenize each bulk material with a mincer and weigh 2-g test portions into 50 mL screw cap tubes for the Biofish methods. 2.5.3 For each collaborator, randomize and blind code two test portions of each material. 2.5.4 Bag and label the Biofish test portion set for each collaborator. 2.5.5 In addition, weigh six 50-g test portions of Materials 1, 4, and 6 for the OMA 990.28 reference method. 2.5.6 Randomize and blind code two OMA test portions of each material, bag and label the test portion sets for three collaborators. One of the collaborators can be the organizing laboratory. 2.5.7 Freeze all test portions at -80°C. Shipment of Test Portions 2.6.1 Test portions will be packaged in leak-proof insulated containers and shipped on dry ice by overnight carrier to arrive on Tuesday before initiation of analysis .

2.6

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2.6.2 Upon arrival at the collaborative site, the collaborator will open the shipment, examine the contents for leakage, and inspect each test portion to ensure all are frozen. All test portions will be stored at -20°C overnight. 2.7 Analysis of Test Portions by BIOFISH 300 SUL On Wednesday, each collaborator will analyze their test portions as follows. 2.7.1 Refer to the Test Portion Inventory Sheet for the BIOFISH 300 SUL range to use for each test portion. There are 4 blind coded test portions for each method range. Group the test portions by range keeping them frozen. 2.7.2 Starting with the test portions for the first range, for example, 7-30 mg/kg, allow the appropriate test portions to sit at room temperature for no more than 15 minutes to thaw, with the plastic tube closed, as it was received. 2.7.3 Add 18 mL of SUL extraction solution. 2.7.4 Homogenize the contents with the aid of an UltraTurrax set at 8000-9000 rpm for 15-20 sec. 2.7.5 Let stand a minimum of 15 minutes at room temperature to complete the extraction. Once extracted, the samples are stable up to 1 h at room temperature. 2.7.6 Follow the BIOFISH 300 SUL 7-30 mg/kg method to set up the device (recondition, activation and calibration). 2.7.7 Follow the BIOFISH 300 SUL 7-30 mg/kg method to complete the analyses. Analysis of Test Portions by OMA 990.28 – Performed by 3 Collaborators only Beginning on Wednesday, select collaborators will analyze six 50-g test portions by the OMA 990.28 method. Keep each test portion frozen until ready for analysis. Complete the analyses within three days. Briefly: 2.8.1 Prepare apparatus and solutions according to the OMA 990.28 method. 2.8.2 Thaw each test portion just prior to analysis. 2.8.3 Add 100 mL 5% EtOH in water v/v and blend. 2.8.4 Transfer test suspension to prepared apparatus. 2.8.5 Allow all but 2-3 mL of HCl solution from separatory funnel to drip into flask containing test suspension. 2.8.6 Boil contents of flask for 1.7 h. 2.8.7 Titrate contents of vessel with 0.010 M NaOH to a persistent yellow end point. 2.8.8 Calculate SO 2 content. 2.8.9 Record results on the Data Report Form (Appendix 3.5) 2.7.8 Repeat steps 2.7.2 – 2.7.7 for the other two method ranges. 2.7.9 Record all results on the Data Report Form (Appendix 3.5). 2.8

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2.9 Analysis of QC Test Portion Sets 2.9.1 The Coordinating Laboratory will retain two Quality Control Test Portion Sets. These sets are identical to the collaborator test portion sets and will be randomized and blind coded. 2.9.2 QC Set 1 will be analyzed on the day of shipment to check the sulfite levels. These data are used only to gauge whether the target levels will be met in the study. 2.9.3 QC Set 2 will be the “official” test portion set analyzed by the Coordinating Laboratory on the day the collaborators initiate analyses and will follow section 2.7 above. 2.10 Reporting Raw Data 2.10.1 Data will be reported using the data report form in Appendix 3.5. 2.10.2 Each collaborator will complete the data report form and email it to the co- Study Director as indicated on the form. 2.10.3 Each collaborator or collaboration site must retain all related test results for a minimum of one year. 2.10.4 Collaborators may also submit a comment form (Appendix 3.4). 2.10.5 A table reporting all raw data from all collaborators must be included in the collaborative study report. All submitted collaborator comments must be included in the collaborative study report. 2.11 Analyzing Raw Data 2.11.1 The Study Director will analyze the study data once all data report forms have been received. 2.11.2 Construct a Youden plot of replicate pairs by plotting first replicate versus second replicate for each material. Usually major discrepancies will be apparent. 2.11.3 Access the “AOAC Interlaboratory Study Workbook – Blind (Unpaired) Replicates” for statistical analyses: http://www.aoac.org/AOAC_Prod_Imis/AOAC/SD/SGR/AOAC_Member/SDCF/S DGRCF/SDSGM.aspx?hkey=086fdba4-32e2-43f0-8272-7d41a6603e87 2.11.4 Analyze each material and each method separately. Look for significant findings from Cochran’s and Grubbs’ outlier tests. 2.11.5 Data may be excluded due to an assignable cause if sufficient justification is provided. Data may not be excluded on a statistical basis only. 2.11.6 Using the valid data, report the following parameters for each material and each method: 2.11.6.1 Mean Concentration .—Mean candidate method result across all collaborators.

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2.11.6.2 Standard Deviation of Repeatability (s r

) .-Calculate repeatability as the

standard deviation of replicates at each concentration for each laboratory.

2.11.6.3 Relative Standard Deviation of Repeatability (RSD r ] x 100. 2.11.6.4 Standard Deviation of Reproducibility (s R [s r /mean cand

) .—RSD r

=

) .-Calculate reproducibility as the standard deviation of replicates at each concentration across all collaborators.

2.11.6.5 Relative Standard Deviation of Reproducibility (RSD R

). —RSD r

=

[s r

/mean cand

] x 100.

2.12 Collaborative Study Report Follow the JAOACI Author Instructions [ http://www.aoac.org/aoac_prod_imis/AOAC_Docs/Journal/Author_Resources.pdf ] to prepare a manuscript in the AOAC Style. 3.0 Appendices

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Appendix 3.1

Instructions to Collaborators Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study

General Instructions about Collaborative Studies Introduction

The purpose of this document is to provide detailed instructions for performing the collaborative study for the BIOFISH 300 SUL method. Study Director Sandra Salleres will conduct the study. The Study Director is responsible for providing method-specific training, providing test portions, clarifying procedures, collating results, and submitting a report to AOAC INTERNATIONAL. Should any questions arise before or during the course of the study, please direct them immediately to the Study Director: Sandra Salleres

Product Development Manager Biolan Microbiosensores, S.L. Parque Tecnológico de Bizkaia, Laida Bidea 409 Zamudio, Bizkaia 48170 Spain Tel.: +34 946 574 161 ssalleres@biolanmb.com

Important Information 1. Read the method carefully. If you have any questions, contact the Study Director.

2. It is advised to make at least one practice run before the trial using your own materials so that you can minimize errors in manipulations. Check that all pipettes, equipment, and supplies are on hand and calibrated. 3. Begin the analyses on the specified date. Store the test portions according to the instructions. It is essential for the validity of the trial that all collaborators commence the analysis of each test portion on the designated day. Immediately upon receiving the shipment, confirm the contents with the Study Director by email. 4. THIS IS A STUDY OF THE METHOD, NOT OF THE LABORATORY. THE METHOD MUST BE FOLLOWED AS CLOSELY AS PRACTICABLE, AND ANY DEVIATIONS FROM THE METHOD AS DESCRIBED, NO MATTER HOW TRIVIAL THEY MAY SEEM, MUST BE NOTED ON THE REPORT FORM. 5. Report all of your results as soon as analyses are completed. Do not do more or less than indicated in the instructions. For example, do not do duplicate analyses and report the best or average result. More or fewer results complicate the statistical analyses and may invalidate your results. Data sheets are provided with instructions and indicate which results to report. A space is provided on the Data Report Form to indicate any deviations to the methods or deviations to the instructions to

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collaborators. Please include any criticisms, suggested improvements, or general comments about the products on the Collaborators’ Comments Form provided. Any results that were derived from modified protocols should be included but must be clearly indicated. Results and comments should be returned to the Study Director immediately upon completion of the study. Information about this Collaborative Study Shipment Schedule and Receipt of Study Materials and Test Portions Equipment and test kits should arrive in the weeks prior to the prearranged training date for your site. Study materials (test portions) should be received by Tuesday of Week 1. If all collaborators are not on- site at the time of arrival, a designated site coordinator shall receive the materials. Collaborators or site coordinators shall inventory all equipment, test kits, and study materials against the packing lists upon arrival to ensure all materials have arrived. Contact the Study Director immediately if any equipment or test kits have not arrived prior to your prearranged training date or if study materials have not arrived by Tuesday prior to initiation of the study. Test portions will be shipped by overnight courier on Monday of Week 1 to arrive on Tuesday. If all collaborators are not on-site at the time of arrival, a designated site coordinator shall receive the packages. Each package shall be examined for potential damage. The collaborator or site coordinator must alert Study Director if any packages appear compromised or if the contents do not appear frozen. Each collaborator should receive 1 set of 12 blind coded test portions (2 g) for BIOFISH 300 SUL analysis. Select collaborators will receive a second set of 12 blind coded test portions (50 g) for AOAC Official Method 990.28 analysis. Inventory the test portions against the test portion inventory sheet included in the shipment to be sure all are present and alert the Study Director if any test portions are missing. The test portions should be stored at -20°C until initiation of the study on the following day (Wednesday). Table 1. Study Schedule (Specific Dates TBD)

Receipt of BIOFISH 300 SUL

Submission of Data Report Form Week 2, Wednesday

Collaborator Activity

Receipt of Test Portions

Initiation of Analyses Week 1, Wednesday

Training

Week 1, Tuesday

Date

TBD

TBD

Safety Precautions KITCFSUL/KITVFSUL: Not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008. This substance is not classified as dangerous according to Directive 67/548/EEC. 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.

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KITMFSUL/SEFSUL: According to Regulation (EC) No. 1272/2008:

Hazard Statements : H319. Eye Irrit.2. May cause serious eye irritation. Precautionary Statements :

P264 Wash thoroughly after handling. P280 Wear eye protection/face protection. P305+P351+P338 In case of eye contact: rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. P337+P313 If eye irritation persists, get medical advice. Test Portion Analysis On Wednesday, all collaborators shall initiate the extraction and analysis of test portions according to the BIOFISH 300 SUL method (Appendix 3.3). A copy of the detailed method will be provided for you. It is important that all collaborators perform the study on the same day and complete all the testing within one day. All test portions are pre-weighed. Each collaborator will analyze their test portions as follows. 1. Refer to the Test Portion Inventory Sheet for the BIOFISH 300 SUL range to use for each test portion. There are 4 blind coded test portions for each method range. Group the test portions by range keeping them frozen. 2. Starting with the test portions for the first range, for example, 7-30 mg/kg, allow the appropriate test portions to sit at room temperature for no more than 15 minutes to thaw, with the plastic tube closed, as it was received. 3. Add 18 mL of SUL extraction solution. 4. Homogenize the contents with the aid of an UltraTurrax set at 8000-9000 rpm for 15-20 sec. 5. Let stand a minimum of 15 minutes at room temperature to complete the extraction. Once extracted, the samples are stable up to 1 h at room temperature. 6. Follow the BIOFISH 300 SUL 7-30 mg/kg method to set up the device (recondition, activation and calibration). 7. Follow the BIOFISH 300 SUL 7-30 mg/kg method to complete the analyses. 8. Repeat steps 2 –7 for the other two method ranges. 9. Record all results on the Data Report Form (Appendix 3.5). Select collaborators, as prearranged, will receive a set of 6 blind coded test portions for the OMA 990.28 method. Analyze these test portions also beginning on Wednesday as follows. Keep each test portion 2. Thaw each test portion just prior to analysis. 3. Add 100 mL 5% EtOH in water v/v and blend. 4. Transfer test suspension to prepared apparatus. 5. Allow all but 2-3 mL of HCl solution from separatory funnel to drip into flask containing test suspension. 6. Boil contents of flask for 1.7 h. frozen until ready for analysis. Complete the OMA analyses within 3 days. 1. Prepare apparatus and solutions according to the OMA 990.28 method.

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7. Titrate contents of vessel with 0.010 M NaOH to a persistent yellow end point. 8. Calculate SO 2 content. 9. Record results on the Data Report Form (Appendix 3.5) Reporting Results Complete the Data Report Form (included in your packet as Excel file Appendix 3.5), noting any deviations. Once completed, electronically sign and date the form, indicating your QC check of the data, and submit it by email to the Study Director. Note that all collaborators will complete the BIOFISH 300 SUL tab of the workbook, but only preselected collaborators will complete the OMA 990.28 tab of the workbook. Collaborator Comment Form At the completion of the study, please fill out the Collaborator Comment Form (Appendix 3.4) to submit any comments you may have regarding the BIOFISH 300 SUL method or the conduct of the study. Completed forms should be returned by email to the Study Director.

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Appendix 3.2

Study Materials and Apparatus Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study

Materials provided by collaborator:

Apparatus (standard lab apparatus): (a) Tissue homogenizer .--UltraTurrax IKA T-25 or equivalent. (b) Pipets.-- Adjustable, capable of delivering 20-200 μL and 500-5000 μL. (c) Graduated cylinder.-- 25 mL. (d) Computer. —Windows-based PC with USB port to connect to device.

Consumables: (a) Pipet tips . (b) Distilled water.

Materials provided by Biolan Microbiosensores:

(a) BIOFISH 300 Biosensor .-- BF300-004 (b) BIOFISH .-- BTFSUL100 and BTFSULAL100 (c) SUL CALIBRATION KIT (KITCFSUL)

(d) SUL MEASUREMENT KIT (KITMFSUL1 or KITMFSUL2) (e) SUL EXTRACTION SOLUTION (SEFSUL 1 or SEFSUL2) (f) Draft OMA Method .-- (g) BIOFISH sample set .--1 set of 12 blind coded tubes per collaborator packaged in 50 mL conical tubes and frozen. (h) OMA sample set.-- 1 set of 6 blind coded tubes for select collaborators packaged in 50 mL conical tubes and frozen.

Collaborators preselected to perform the OMA 990.28 reference method will provide all necessary materials, reagents, and apparatus to perform the method.

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Appendix 3.3

AOAC Official Method XXXX.XX Determination of Sulfites in Shrimp

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 Table XXXX.XXA for a summary of results of the collaborative study, supporting acceptance of the method. See Tables X-Y for detailed results of the collaborative study [ 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). ( c ) Mincer.--

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( d ) Sample homogenizer.-- Ultraturrax® or equivalent. (e) Adjustable pipets.-- Capable of delivering 20-200 μL and 500-5000 μL.

D. Additional Supplies and Reagents ( a ) Distilled water.-- ( b ) Plastic tubes. --50 mL. ( c ) Graduated cylinder.– 25 mL. ( d ) Pipet tips . --

( e ) Bottles with caps. —1- and 2-L sizes. (f) Liquid dispenser.--

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 μL 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 μL 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 μL 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 μL 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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Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

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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Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

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 accoustic 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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Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

Appendix 3.4

Collaborator Comment Form

Collaborator:

Site:

Comments about the Method:

Comments about the Conduct of the Study:

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Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

Appendix 3.5

See attached file “BIOFISH 300 SUL Data Report Form.xlsx”

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Collaborative Study Data Report Form - BIOFISH 300 SUL - All Collaborators

Raw and Boiled Shrimp - 2 g Test Portions

Fill in shaded areas as appropriate.

Site: Collaborator: BIOFISH Biotest Lot #: SUL Calibration Kit Lot #

SUL Measurement Kit Lot # SUL Extraction Solution Lot # Reference Electrode Lot # Electrode Storage Solution Lot # SUL Verifying Kit Lot # Analysis Start Date: Analysis Completion Date:

Test Portion ID

Sulfite, mg/kg

Note any deviations here:

Data QC'd by Collaborator:

Name

Date

When completed and QC'd, email workbook to ssaleres@biolanmb.com

Collaborative Study Data Report Form - OMA 990.28 - Select Collaborators Only

Raw and Boiled Shrimp - 50 g Test Portions

Fill in shaded areas as appropriate.

Site: Collaborator: Analysis Start Date: Analysis Completion Date:

Test Portion Weight, mg

Volume of 10 mM NaOH (mL)

Sulfite Content, µg/g

Test Portion ID

Note any deviations here:

Data QC'd by Collaborator:

Name

Date

When completed and QC'd, email workbook to ssaleres@biolanmb.com

47.3.43

PO Box 1178, Portsmouth, NH 03801, USA), or equivalent, is suitable. C. Reagents ( a ) Aqueous hydrochloric acid .—4M. For each analysis, prepare 90 mL solution by adding 30 mL HCl to 60 mL deionized (18 megohm) water. ( b ) Methyl red indicator .—Dissolve 250 mg methyl red in 100 mL ethanol. ( c ) Standardized titrant .—0.010MNaOH. Certified reagent may be used (Fisher SO-5-284). Standardize solution with reference standard potassium acid phthalate. ( d ) Hydrogen peroxide solution .—3%. For each analysis, dilute 3 mL ACS reagent grade 30% H 2 O 2 to 30 mL with deionized (18 megohm) water. Just prior to use, add 3 drops methyl red indicator and titrate with 0.010M NaOH to yellow end point. If end point is exceeded, discard solution. ( e ) Nitrogen .—High purity, used with regulator to maintain flow of 200 mL/min. To guard against oxygen in N 2 gas, use GC-type trap (Oxy-Purge N [Alltech-Applied Science Laboratories, Inc.; www.alltechweb.com], or equivalent). Alternatively, oxygen-scrubbing solution, such as alkaline pyrogallol, in gas-washing bottle (Kimble Glass, Inc.) may be used.

AOAC Official Method 990.28 Sulfites in Foods Optimized Monier–Williams Method

First Action 1990 Final Action 1994

(Applicable of determination of ³ 10 ppm ( m g/g) sulfites in foods. Applicable in presence of other volatile sulfur compounds; not applicable to dried onions, leeks, and cabbage.) See Table 990.28 for the results of the interlaboratory study supporting acceptance of the method. A. Principle Method measures free sulfite plus reproducible portion of bound sulfites, such as carbonyl addition products, in foods. Test portion is heated with refluxing HCl (ca 1M) to convert sulfite to SO 2 . Stream of N 2 introduced below surface of refluxing solution sweeps SO 2 through water-cooled condenser and, via bubbler attached to condenser, with 3%H 2 O 2 solution, where SO 2 is oxidized to H 2 SO 4 . Sulfite content is directly related to generated H 2 SO 4 , which is determined by titration with standardized NaOH solution. For verification, sulfate can be determined gravimetrically as BaSO 4 . B. Apparatus ( a ) Distillation apparatus .—( Note: In this method, back pressure inside apparatus is limited to unavoidable pressure due to height of 3% H 2 O 2 solution above tip of bubbler (F). Keep back pressure as low as possible to avoid loss of SO 2 through leaks. Use thin film of stopcock grease on sealing surfaces of all joints except joint between separatory funnel and flask. Clamp together each joint to ensure complete seal throughout analysis.) Assemble apparatus (Figure 990.28A ), which includes ( 1 ) inlet adapter (A) with hose connector (Kontes 183000). Adapter provides means of applying head pressure above solution. Use of pressure-equalizing dropping funnel is not recommended because condensate, perhaps containing SO 2 , is deposited in funnel and side arm. ( 2 ) Separatory funnel (B), ³ 100mLcapacity. ( 3 ) Round-bottom flask (C), 1 L, with three 24/40 tapered joints. ( 4 ) Gas inlet tube (D) (Kontes 179000) of sufficient length to permit introduction of N 2 within 2.5 cm of bottom of flask. ( 5 ) Allihn condenser (E) (Kontes 431000-2430), jacket length 300 mm. ( 6 ) Bubbler (F), fabricated from glass according to dimensions in Figure 990.28B . ( 7 ) Vessel (G), ca 2.5 cm id and 18 cm deep. ( b ) Buret .—10 mL (Kimble Glass, Inc., No. 17124-F) with overflow tube and hose connections for Ascarite tube or equivalent air-scrubbing apparatus to permit maintenance of CO 2 -free atmosphere over standardized 0.010M NaOH. ( c ) Chilled water circulator .—Chill condenser with coolant, such as methanol–water (20 + 40, v/v), maintained at £ 15°C. Circulating pump, Neslab Coolflow 33 (Neslab Instruments, Inc.,

Table 990.28. Interlaboratory study results for sulfites in foods

Mean, m g/g s r

RSD r

, % s R

RSD R

, % HorRat

Matrix

Figure 990.28A. Apparatus for optimized Monier-Williams method: A, inlet adapter; B, separatory funnel; C, round-bottom flask; D, gas inlet tube; E, Allihn condenser; F, bubbler; G, vessel.

Hominy

9.17 1.33 14.5 1.42 15.5 8.05 1.36 16.9 1.62 20.1

1.36 1.73 2.38

Fruit juice

Protein (seafood)

10.41

1.47 14.1 2.77 26.6

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Use rubber bulb equipped with valve to apply head pressure above HCl in separatory funnel. Open stopcock in separatory funnel and let HCl flow into flask. Continue to maintain sufficient pressure above acid solution to force solution into flask. Stopcock may be closed, if necessary, to pump up pressure above acid, and then opened again. Close stopcock before last 2–3 mL drain out of separatory funnel to guard against escape of SO 2 into separatory funnel. Apply power to heating mantle. Use power setting that causes 80–90 drops/min of condensate to return to flask from condenser. Let contents of flask boil 1.7 h, and then remove vessel (G). G. Determination ( a ) Titration .—Immediately titrate contents of vessel (G) with 0.010M NaOH to yellow end point that persists ³ 20 s. Compute sulfite content, expressed in m g SO 2 /g food (ppm), as follows: where 32.03 = milliequivalent weight of SO 2 ; V B = volume (mL) of NaOH of molarity M required to reach end point; 1000 = factor to convert milliequivalents to microequivalents; weight = weight, g, of test portion introduced into 1 L flask. ( b ) Gravimetric determination .—Optional. Following titration, rinse contents of vessel (G) into 400 mL beaker. Add 4 drops 1M HCl and excess of filtered 10%BaCl 2 solution, and let mixture stand overnight. Wash precipitate by decantation 3 times with hot water through weighed Gooch crucible. Wash with 20 mL alcohol and 20 mL ether, and dry at 105 ° –110 ° C. ( c ) Blank determination .—Determine blank on reagents both by titration and gravimetrically, and correct results accordingly. H. Recovery Assays To become familiar and proficient with method before routine use, analyze foods containing known amounts of sulfite. Perform analysis in manner that precludes any loss of sulfite by oxidation or reaction with components in food. Since sulfites are reactive with air and food matrixes and lack stability, fortify portions with stable source of sulfite, not sodium sulfite or similar salts. Sodium hydroxymethylsulfonate (HMS), which is bisulfite addition product of formaldehyde and is structurally similar to some combined forms of sulfite in foods, is useful for preparing stable fortified test materials. For analysis, transfer 50 g prepared test portion of sulfite-free food to Monier-Williams flask. Add aliquot of aqueous solution of HMS sodium salt. Analyze solution immediately. HMS recoveries of ³ 80% from food matrixes fortified at 10 m g/g are recommended to ensure accurate analytical data. SO 2 , m g/g (ppm ) = 32 03 1000 . ´ ´ ´ V M weight B SO 2 , m g/g (ppm) = mg BaSO g test portion 4 ´ 274 46.

Figure 990.28B. Enlarged diagram of bubbler for Monier-Williams apparatus (lengths in mm).

Prepare trap as follows: ( 1 ) Add 4.5 g pyrogallol to trap. ( 2 ) Purge trap with N 2 for 2–3 min. ( 3 ) Prepare KOH solution by adding 65 g KOH to 85 mL H 2 O. ( Caution: Heat is generated.) ( 4 ) Add KOH solution to trap while atmosphere of N 2 is maintained in trap. D. Preparation of Test Suspension ( a ) Solids .—Transfer 50 g food, or quantity that contains 500–1500 m g SO 2 , to food processor or blender. Add 100 mL ethanol–water (5 + 95, v/v) and briefly grind mixture. Continue grinding or blending only until food is chopped into pieces small enough to pass through standard taper 24/40 joint of flask (C). ( b ) Liquids .—Mix 50 g test portion, or quantity that contains 500–1500 m g SO 2 with 100 mL ethanol–water (5 + 95, v/v). ( Note: Carry out test suspension preparation and analysis as quickly as possible to avoid loss of labile forms of sulfite.) E. System Preparation Using apparatus assembled as shown in Figure 990.28A , position flask (C) in heating mantle controlled by power-regulating device (rheostat), and add 400 mL H 2 O to flask. Close stopcock of separatory funnel (B) and add 90 mL 4M HCl to separatory funnel. Begin N 2 flow at 200 ± 10 mL/min. Initiate condenser coolant flow at this time. To vessel (G) add 30 mL 3% H 2 O 2 , which has been titrated to yellow end point with 0.010M NaOH. After 15 min, apparatus and water will be thoroughly deoxygenated and prepared Remove separatory funnel (B) and quantitatively transfer test suspension in aqueous ethanol to flask (C). Wipe tapered joint clean with laboratory tissue, quickly apply stopcock grease to outer joint of separatory funnel, and return separatory funnel to flask. Nitrogen flow through 3% H 2 O 2 solution resumes as soon as separatory funnel is reinserted into appropriate joint in flask. Examine each joint to be sure that it is sealed. test suspension may be introduced into system. F. Suspension Introduction and Distillation

Reference: JAOAC 72 , 470(1989). CAS-7446-09-5 (sulfur dioxide)

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