AOAC RI ERP E-Book - DS DF

OMA 2014.10 C: Collaborative Study Manuscript Expert Review Panel Use Only September, 2017

2013 Dietary Starch in Animal Feeds Collab Study Protocol 072513

2. Method Performance Parameters (based on the research published in JAOACI) REPEATABILITY The standard deviations of replicates for dietary starch analysis of the food and feed substrates and for purified substrates were low did not differ among assays (Tables 1 and 2) ( P > 0.36). These values are comparable to or less than repeatability standard deviation values (1.6 to 2.2) previously reported for method the extended AOAC Method 996.11 when food or feed samples were analyzed (2). Repeatability of the glucose measurement assay used for the dietary starch method based on absorbance values on triplicate samples of standard solutions was very good within run and is a key reason that the small deviations from linearity could be detected. For the glucose standards prepared in benzoic acid, the 0.1:3.0 standard solution : GOPOD reagent ratio gave standard errors of 0.0022, 0.0012, 0.0018, 0.0021, and 0.0030 for 0, 250, 500, 750, and 1000 µ g glucose/mL standards (overall coefficient of variation % glucose-containing solutions = 0.31%). Values for the freshly prepared samples were similar. LIMIT OF DETERMINATION Limit of determination for the dietary starch assay based on absorbance values of undiluted reagent blanks was calculated as mean blank value + 3 x blank standard deviations (3). Based on 1:1 to 1:3 dilution of reagent blanks converted back to an undiluted basis, and including results from assays performed outside of this study (5 blanks with 2 readings each from 5 separate assay runs) the mean absorbance + standard deviation of undiluted blanks was 0.016 + 0.004 for a detection limit of 0.028 absorbance. Using average values obtained for standard curves, starch detection limits are 0.2% of sample dry matter based on a 100 mg sample of 90% dry matter that required no dilution at the final volume for each assay. Limits of determination for glucose measurement were calculated from absorbance values of 0 µ g glucose / mL standards analyzed in triplicate from 4 assay runs in which the glucose standards were prepared in 0.2% benzoic acid solution. Values were calculated as mean blank value + 3 x blank standard deviations (3). For the 0.1:3.0 standard solution: GOPOD reagent samples, the mean absorbance + standard deviation of undiluted blanks were 0.0002 + 0.0020 for a detection limit of 0.0063 absorbance. Using average quadratic standard curves, the calculated glucose detection limit is 5.12 µ g/mL for 0.1:3.0 standard solution: GOPOD. The detection limit represents approximately 0.5% of the range of the glucose standards. greatest for the candidate assay ( P = 0.05), and did not differ between the hot water and extension of the AOAC method (Table 4). Recovery of corn starch using 200 units of amyloglucosidase and determined with samples used for the dietary starch assay linearity evaluation runs was 99.7 + 1.4% which is comparable to that of the AOAC Method 996.11 of (99.7%, maximum: 111.75%, minimum: 87.24%, number of labs: 50, maize starch run in duplicate; table 1 in paper, results for maize starch divided by 100% minus 13.35% moisture content to give values on a dry matter basis)(2). AOAC Research Institute ERP Use Only ACCURACY / RECOVERY Starch recovery values from purified starch substrates in the preliminary study were

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