Analysis of Sugars in Feeds by HPLC with Post-column Derivatization and Fluorescence Detection

The types and amounts of sugar in animal feeds are as important as the amount of protein, minerals, and fats in the determination of nutritive value. We developed a simple and sensitive HPLC method for analyzing six sugars in animal feeds – sucrose, fructose, glucose, galactose, maltose, and lactose. Post-column derivatization reagents convert reducing and nonreducing sugars into fluorescent derivatives, which greatly improves the sensitivity and selectivity of the detection.

Figure 1: Chromatogram of a standard solution of sugars. Fructose 500 ppm, glucose 500 ppm, galactose 500 ppm, sucrose 3000 ppm, maltose 500 ppm, and lactose 500 ppm.

The blends of feed examined varied from grains/vegetable products (live stock feeds) to meat/vegetable products (pet food).

Figure 2: Chromatogram of a feed sample. Levels of sugars present in the sample: fructose 0.54%, glucose 0.52%, galactose 0.09%, sucrose 4.02%, maltose 1.12%.

Method

Sample Preparation

Mix 2.5 g of feed sample with 50 mL of water. Heat using a water bath while constantly mixing for 1 h at 65 °C. Centrifuge and filter through 0.45 μm filter.

Calibration

A quadratic calibration curve with correlation > 0.999 is observed for monosaccharides such as fructose, glucose, and galactose. A linear calibration curve with correlation > 0.999 is observed for disaccharides such as maltose, lactose, and sucrose.

Figure 3: Chromatogram of a feed sample spiked with sugars. Total levels for sugars: fructose 1.14%, glucose 1.52%, galactose 0.69%, sucrose 6.02%, maltose 1.72%, lactose 0.6%.

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