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OMA 2014.10 B: JAOAC Article Expert Review Panel Use Only September, 2017

404  H all : J ournal of AOAC I nternational V ol . 98, N o . 2, 2015

( 13 ) Set spectrophotometer tomeasure absorbance at 505 nm. After the incubation is complete, zero the spectrophotometer with the GOPOD-reacted 0 µg/mL working standard solution. Read absorbances of remaining GOPOD-reacted working standard solutions, and reagent blank, control sample, and test sample solutions. All reacted solutions must be read within 30 min of the end of the GOPOD incubation. The duplicate absorbance values are averaged for each reagent blank, test sample, and control sample solution and used in Calculations . F. Calculations Determine the quadratic equation that fits the absorbances of the working standard solutions. The absorbance values, A CF or A CE , are the independent variables (X), and actual glucose concentrations are the dependent variables (Y). Individual absorbance values of the working standard solutions, not averages, are used. The equation has the form: µg Glucose/mL = ( A CF or CE 2 × Q + A CF or CE × S + I ) Calculate dietary starch content in test sample as received as follows: Free glucose, % = ( A CF 2 × Q + A CF × S + I ) × V F × DF F × 1/1000000 × 1/ W F × 162/180 × 100 × 1/ W E × 162/180 × 100] – 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 two replicates for each test solution; Q = quadratic slope term, S = linear slope term, and I = intercept of the standard curve to convert absorbance values to µg glucose/mL; V F , V E = final sample solution volume, ca 50.0 mL for V F and 51.1 mL for V E if done by summation of volumetric additions, otherwise, by size of volumetric flask used; DF = dilution factor, e.g., 0.5 mL sample solution diluted into 5.0 mL = 5.0/0.5 = 10; 1  g/1000000 µg = conversion from µg 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. If test samples are run in duplicate portions, the free glucose % in the dietary starch equation is the average free glucose % value determined for the test sample. Evaluation of the Dietary Starch Method Initial evaluation of data from all laboratories showed that most outliers occurred in two laboratories (Table 2). Laboratory 14 had significant Cochran’s tests for five of the test materials, indicating suspect replicate results within this laboratory. Unlike the other laboratories, Laboratory 14 ran duplicate portions of test materials on separate days, rather than together within the same run. Based on laboratory ranking scores (18), this laboratory was designated as an outlier and its data were Results and Discussion Dietary starch, % = [( A CE 2 × Q + A CE × S + I ) × V E × DF E × 1/1000000

W E : Samples for Enzymatically-Released + Free Glucose Analysis

W F : Samples for Free Glucose Analysis

Test and Control Sample Portions and Blanks

Test and Control Sample Portions and Blanks

Add 30 mL Na acetate buffer and heat-stable, alpha-amylase.

Add 30 mL Na acetate buffer

Vortex. Incubate 1 h at 100°C. Vortex at 10, 30 and 50 min.

Vortex. Incubate 1 h at 100°C. Vortex at 10, 30 and 50 min.

Add 20 ml water, or filter and bring to 100 mL volume in a volumetric flask.

Cool on bench 0.5 h.

Invert tubes >4 x to mix completely.

Add diluted amyloglucosidase.

Vortex. Incubate 2 h at 50°C. Vortex at 1 h. Invert tubes >4 x to mix completely.

Add 20 ml water, or filter and bring to 100 mL volume in a volumetric flask.

Test Solutions

Volume by Sum of Volume Additions Centrifuge portion at 1000 x g for 10 min (if still cloudy, centrifuge 10 min at 10,000 x g ).

Volume Using Volumetric Flasks Proceed to dilution step.

Prepare dilutions as needed or analyze test solutions directly.

In duplicate, pipette 0.1 mL working standards and test solutions into 16 x 100 mm glass tubes, add 3.0 mL GOPOD.

Vortex. cover tubes with plastic film to seal. Incubate in a 50°C waterbath for 20 min.

Solutions with Developed Chromogen

Read absorbance on a spectrophotometer.

Figure 2014.10. Flow chart of the dietary starch assay.

10000 ×  g to clarify the solution before proceeding. Solutions may increase in temperature during centrifugation; allow centrifuged solutions to come to room temperature before preparing dilution. ( b ) Volume using volumetric flasks .—Quantitatively transfer test sample solutions with filtration through a hardened paper filter with 22 µm retention and rinses with water to 100 mL volumetric flasks. ( 10 ) Prepare dilutions as needed with distilled or deionized water. Solutions from control samples and test samples estimated to give greater than 1000 µg glucose/mL concentrations of free and released glucose should be diluted 1 in 10 if processed as in ( 9 )( a ) or 1 in 5 if processed as in ( 9 )( b ). Reagent blanks should be diluted to provide solutions with the same dilutions as used with the test solutions, so that the diluted reagent blank solutions can be used to make corrections for similarly diluted test solutions. Dilutions may be prepared using volumetric flasks or by accurate pipetting. If done by pipetting, use a minimum of 0.5 mL test sample or control solution to minimize the impact of variation in pipetting small volumes. ( 11 ) Pipet 0.1 mL in duplicate of glucose working standard solutions (0, 250, 500, 750, and 1000 µg/mL glucose), C ( f ), and reagent blank, quality control sample, and test sample solutions into the bottoms of 16 × 100 mm glass test tubes using two tubes/solution. Add 3.0 mL GOPOD reagent, C ( 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, C ( e )( 2 ), for measurement of glucose in working standards, reagent blank, control sample, and test sample solutions. ( 12 ) Incubate in a 50°C water bath for 20 min.

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