High performance liquid chromatographic method with post-column detection for quantification of reducing sugars in foods

Abstract

A novel liquid chromatographic analysis method with post-column detection for sugars was developed to improve existing methods in regard to operation time, selectivity, and sensitivity. This method involves separation of reducing sugars on HPLC column at 30 °C and 0.8 mL min−1 flow rate, post−column reaction of sugars with Cu(II)-neocuproine (Nc) reagent at 80 °C and 0.3 mL min−1 flow rate, and measurement of Cu(I)-Nc product at 450 nm. The proposed assay was applied to glucose, fructose, maltose, and lactose as reducing sugars. Non-reducing sucrose was determined indirectly, after conversion to its constitutive monomers glucose and fructose by hydrolysis, and analysis with a relative error from -2.41 to 2.09%. Honey, apple juice, and milk samples were evaluated as commercial products. The results obtained with the proposed assay compared to those of the alkaline Cu(II)−Nc reference method were found close to each other, and compatible with the label values of commercial products. The accuracy of the developed method was performed by spiking glucose to honey and lactose to milk samples using two different concentrations. The obtained recoveries with respect to the post-column HPLC method were between 97 and 105% for honey and 96–107% for milk. The method gave linear responses against sugar concentration with correlation coefficients greater than 0.996 for the four analytes (glucose, fructose, maltose and lactose) in a range of 9.0 - 342.3 mg L−1 with LOD values ≤ 7.4 mg L−1. With the developed method, it was possible to sensitively determine reducing sugars in various food samples at a lower temperature of post-column reaction (compared to literature values) with easy application of low cost reagents requiring minimal preliminary operation.