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The disaccharide lactulose is known to have a large influence on human digestion, the effects of which range from prebiotic to laxative action. Lactulose can be derived from lactose by either alkaline isomerization or enzymatic transgalactosylation of fructose. The aim of this study was to establish a new analytical method for the quantification of both disaccharides lactose and lactulose using high performance liquid chromatography (HPLC) and evaporative light scattering detection (ELSD). The new method needs to provide good chromatographic separation of lactose and lactulose while being fast and reliable. Therefore, four analytical methods using different HPLC columns were developed and compared. A HPLC column with an amino-bonded polymeric matrix yielded better results compared to amino-bonded silica-phase resin or cation-exchange resin. The new method had detection limits for lactose and lactulose of 3.8 and 2.5 mg L −1 and was successfully applied to the quantification of lactulose and lactose in commercial milk products.

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