OMA Protocol Review: OMAMAN-56 Sulfites in Shrimp

Biolan Microbiosensores BIOFISH 300 SUL Collaborative Study Protocol

February 2019

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

3

DRAFT