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

2013 Dietary Starch in Animal Feeds Collab Study Protocol 072513

with each batch of samples. Glucose will allow evaluation of quantitative recovery, and starch will allow evaluation of quantitative recovery and efficacy of the assay.

Preparation of Reagent Blanks, Standard Curves, and Test Samples ( a ) Reagent blank. — For each assay, tubes containing no sample and only the reagents added for each method were carried through the entire procedure. Reagent blanks diluted to the same degree as samples (no dilution or 1 in 10) are analyzed. Absorbance values for the reagent blanks are subtracted from absorbance values of the solutions prepared from test and control samples. ( b ) Standard curves. — Pipette 0.1 mL of 0.2% benzoic acid solution and nominal 250, 500, 750, and 1000 µ g/mL working standard glucose solutions ( f ) in duplicate into the bottom of 16 x 100 mm glass culture tubes. Add 3.0 mL of GOPOD reagent ( e ) to each tube using a positive displacement repeating pipette aimed against wall of tube so it will mix well with the sample. Vortex tubes. Cover tops of tubes with plastic film. Incubate in a 50°C water bath for 20 min. Read absorbance at 505 nm using the 0 µ g glucose/mL standard to zero the spectrophotometer. All readings should be completed within 30 min of the end of incubation; avoid subjecting solutions to sunlight as this degrades the chromogen. Calculate the quadratic equation describing the relationship of glucose µ g/mL (response variable) and absorbance (abs) at 505 nm (independent variable). The equation will have the form quadratic coefficient x abs x abs + linear coefficient x abs + intercept. Use this standard curve to calculate glucose µ g/mL in sample solutions. A new standard curve should be run with each glucose determination run. ( c ) Test samples. — Feed and pet food amenable to drying should be dried at 55°C in a forced-air oven. Dried materials are then ground to pass the 0.5 or 1.0 mm screen of an abrasion mill or the 0.5 mm screen of a cutting mill or other mill fitted with a screen to give an equivalent fineness of grind (to pass a 40 mesh screen). Ground, dried materials are transferred into a wide mouthed jar and mixed well by inversion and tumbling before subsampling. Semi-moist, moist or liquid products may be homogenized, blended or mixed to ensure homogeneity and reduced particle size (10).

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