**4.1 3,5-dinitrosalicylic acid method (Miller, 1959)**

This method was first mentioned by Summer & Sisler (1944) and modified by Miller (1959), with this technique there is a possibility to dosage reducing sugar as total sugar.

The sugar act as a chemical reductor due free aldehyde group or ketone group presence in its molecule. In an alkaline medium, the reducing sugars are able to reduce the 3-5 dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, wherever, the aldehyde group is oxidized to aldonic acid (Figure 2). The 3-amino-5-nitrosalicylic acid is a orange color product, and the intensity of the color depends on the concentration of the reducing sugar. The sodium hydroxide provides the glucose reaction with 3-5-dinitrosalicylic acid by medium alkalinization.

Besides to 3-5-dinitrosalicylic acid, it is also used in this method the Rochelles salt (Potassium sodium tartrate), phenol, sodium bisulfite, and sodium hydroxide. The phenol optimize the quantity of the colour produced and sodium bisulfite stabilize the colour in the phenol presence (Miller, 1959).

The sensitivity of the method is from 100 to 500µg.mL-1 of reducing sugar. A standard glucose or fructose solution is used to build the calibration curve and get the straight line equation to quantify samples.

Fig. 2. Reaction of reducing sugar with 3,5-dinitro-salycilic acid reagent.
