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

can be used to make corrections for similarly diluted test solutions. For (9a) 1 in 10 dilutions and for (9b) 1 in 5 dilutions produced with accurate pipetting or use of volumetric flasks are adequate. ( 11 ) Pipette 0.1 mL in duplicate of working standard solutions (0, 250, 500, 750, and 1000 µ g/mL glucose) (f) , and reagent blanks, quality control samples, and test sample solutions into the bottoms of 16 x 100 mm glass test tubes using 2 tubes/test sample solution. Add 3.0 mL of glucose oxidase-peroxidase (GOPOD) reagent ( e )( 1 ) to each tube. Vortex tubes. Place tubes in a rack and cover with plastic film to seal. Note: Alternative to the use of the GOPOD method, proceed with alternate glucose determination method ( e )( 2 ) for measurement of glucose in working standards, reagent blanks, control samples, and test sample solutions. ( 12 ) Incubate in a 50°C water bath for 20 min. ( 13 ) Set spectrophotometer to measure absorbance at 505 nm or as appropriate for an alternate GOPOD method. Do not allow solutions to be subject to direct sunlight. Zero the spectrophotometer with the 0 µ g / mL working standard solution. Read absorbances of remaining working standard solutions, and solutions from reagent blanks, control samples, and test sample solutions. All solutions must be read within 30 min of the end of the GOPOD incubation. The duplicate values are averaged for each reagent blank, test sample, and control sample solution and used in Calculations . Calculations Determine the quadratic equation of the standard curve that fits the absorbances of the working standard solutions. The absorbance values are the independent variable (X axis), and actual glucose concentrations of the working standard solutions are the dependent variable (Y axis). Individual absorbance values of the working solutions, not averages, are used. Calculate dietary starch content in test samples as received as follows: Free glucose, % = ( A CF 2 x Q + A CF x S + I ) x V F x DF F x 1/1000000 x 1/ W F x 162/180 Dietary Starch, % = [( A CE 2 x Q + A CE x S + I ) x V E x DF E x 1/1000000 x 1/ W E x 162/180] – Free Glucose% Where: subscript F represents values for samples analyzed for free glucose, and subscript E represents values for samples treated with amylase and amyloglucosidase, A CF , A CE = absorbance of reaction solutions minus the absorbance of the appropriately diluted reagent blank, values are averages of the 2 replicates for each test solution; Q = the quadratic slope term, S = the linear slope term and I = the intercept of the standard curve to convert absorbance values to µ g glucose; V F , V E = final sample solution volume; DF = dilution factor e.g., 0.5 mL sample solution diluted into 5.0 mL = 5.0/0.5 = 10; 1g/1000000µg = conversion from µ g to g; W E , W F = test portion weight, as received; 162/180 = factor to convert from measured glucose as determined, to anhydroglucose, as occurs in starch. AOAC Research Institute ERP Use Only

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