RI-ERP-FINALACTION-Recommendations

OMAMAN-20 C/ In House Validation Report ERP Use Only - March 2015

Figure 4 shows the POD response curve which was used to calculate the POD95% concentration. Using a 4-parameter curve fitting a POD95% concentration of 1.5 µg gliadin/100 cm 2 was calculated.

4.2 Independent Validation Studies

External lab not applicable; AACC report (publication in Cereal Foods World ) for collaborative test in preparation by Katharina Scherf (nee Konitzer) and Markus Lacorn.

5 Discussion The immuno-chromatographic dip-stick RIDA®QUICK Gliadin investigated in this validation study was proven to be applicable for the detection of traces of gliadin in a wide variety of foods and on surfaces. The in-house validation included a target and non-target compound study, a lot-to-lot comparability and stability testing, a characterization of ruggedness and a matrix study for non-processed and processed samples. Since a collaborative study with 18 independent labs preceded this in-house validation study, a testing by an independent lab is not necessary. The claimed target prolamins (gliadin, secalin, and hordein) were shown to react in a comparable manner as the “reference” WGPAT gliadin preparation. The 68 non-target compounds (oats, pseudocereals, vegetables, seeds, nuts, fruits, spices, and alternative protein sources as egg, mlik and soy) were checked to be gluten-free (<5 mg gluten/kg) with the R5 sandwich ELISA (AOAC OMA first action 2012.01). The subsequent analysis with the dip- stick revealed no positive results which proved the selectivity of the method. A thorough ruggedness testing included the analysis of variation of ethanol extraction time (30 s and 30 s plus 10 min), incubation temperature of extraction and dip-stick analysis (16°C, 23°C, 30°C), incubation time of the dip-stick (4 min, 5 min, 6 min), and Lot-to-Lot comparison (shelf life and samples). Except incubation times, no parameter was found to influence the result in a way that could be critical under practical conditions. The longer the incubation time the higher the probability of detection for a given concentration was. Therefore, the test kit insert clearly recommends to incubating the dip-stick for exactly 5 min. Nevertheless, this effect is only visible at very low concentrations and not at the legal threshold. As stated by the manufacturer, the shelf life is 18 months at minimum and all tested lots were comparable even when using spiked samples after ethanol or Cocktail extraction. The matrix study can be divided into three parts, (1) for non-processed matrices using the ethanol extraction, (2) for processed matrices which makes the use of the Cocktail solution necessary, and (3) for surfaces. (1) A non-processed naturally contaminated corn flour showed 100% positive results for a concentration around 1 mg/kg, while spiked samples revealed a rate of 100% positive outcomes at 2 mg/kg (rice flour) and 1 mg/kg (soya flour). Due to the uniformity of the results, calculation of a POD 95% concentration was not applicable. (2) For processed samples the concentrations with 100% positive results were 2.2 mg/kg (naturally contaminated cookie) and 4 mg/kg (spiked cookie and spiked sausage). Due to the uniformity of the results, calculation of a POD 95% concentration was not applicable. (3) A stainless-steel surface contaminated with PWG gliadin showed a POD 95% concentration of 1.5 µg/100 cm 2 . The extent of variation within one group between different matrices is very small and can be related to matrix-specific flow behaviors.

AOAC Research Institute Expert Review Panel Use Only

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RIDA®QUICK Gliadin Validation report 2015-01-14