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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Publications at this Location » Publication #232828

Title: Factors Affecting Accuracy and Time Requirements of a Glucose Oxidase-Peroxidase Assay for Determination of Glucose

item Hall, Mary Beth

Submitted to: Journal of Association of Official Analytical Chemists International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2008
Publication Date: 1/15/2009
Citation: Hall, M.B., Keuler, N.S. 2009. Factors Affecting Accuracy and Time Requirements of a Glucose Oxidase-Peroxidase Assay for Determination of Glucose. Journal of Association of Official Analytical Chemists International. 92:50-60.

Interpretive Summary: Analysis of glucose is used for measuring free glucose or glucose as part of starch. The accuracy of glucose and starch values is important for knowing the nutritional value of the foods or feeds. If glucose analysis took less time to perform, more samples could be analyzed, which could increase the throughput of samples in labs and potentially reduce their costs. We evaluated a colorimetric glucose oxidase-peroxidase method for glucose and found that its accuracy could be substantially improved by using a curved (quadratic) rather than straight (linear) equation calculated from the results used to determine glucose content of unknown samples. To this point, linear equations have been used almost exclusively. The assay was reliable to measure glucose in samples with low levels of antioxidants, but foods with higher levels, such as beets, should not be analyzed for glucose with this method. We found that we could reduce the time it took to analyze samples for glucose by about 40% (40 min) and still get good results by modifying the procedure. By improving the accuracy and speed of the glucose assay and determining appropriate applications, the results of the modified analysis can give people a more truthful assessment of the carbohydrate content of foods and feeds. These values can be used to formulate diets to better meet the needs of people and animals.

Technical Abstract: Accurate and rapid assays for glucose are desirable for analysis of glucose and starch in food and feedstuffs. An established colorimetric glucose oxidase-peroxidase method for glucose was modified to reduce analysis time, and evaluated for factors that affected accuracy. Time required to perform the assay was reduced by approximately 40% by decreasing incubation time and removing steps that did not affect absorbance measures. Although linear regressions of absorbance and glucose concentrations of standard solutions exceeded R^2 of 0.9997, evaluation of sum of squared residuals, root mean squared error, and significance of the quadratic term indicated that the curves were quadratic in form. Mutarotatory equilibration of glucose anomers did not appear to be the issue. Evaluation of historic data suggest that the response curve is inherently quadratic. Quadratic curves predicted glucose contents of standard solutions more accurately than linear forms; overestimations at the midpoint of the curve were 0.07, 0.85, and 1.77% for quadratic and linear equations calculated from 5 standard solutions and a linear equation calculated from the 0 and most concentrated standard solution, respectively. Hydrophilic antioxidants at levels no greater than 10 micromole ascorbic acid/0.10 g of air-dried sample did not affect absorbance values.