Dear Suhaila,

Actually what you wrote is exactly right. It is better to use other methods. The following text describes some other methods. For more details and other methods I am attaching the original publication.

5. Fructose determination

5.1 Sulphuric acid-Cysteine-Tryptophan method (Messineo & Edward Musarra,1972) Fructose is dehydrated in acid medium for formation of furfural derivative, which complexs with cysteine hydrochloride to produce a chromophore at starting of green color that is unstable. Immediately, this first chromophore formed after react with tryptophan hydrochloride forming now a chromophore of pink colour that has greater stability chemical than the first and stability of 48 hours.

The reaction is also sensitive because can detect a low quantities like 1µg of fructose. The reaction also is specific because aldohexoses, aldopentoses, and ketopentoses do not interfere, however these compounds do not react even if its concentrations are higher, as 5mg.mL-1. The sensitivity of the method is from 1 to 50µg of fructose. A standard solution of fructose is used to build the calibration curve and to get the equation of the linear regression to quantity samples.

5.1.1 Methodology

Reagents: sulphuric acid 75%, cysteine hydrochloride 2.5%, tryptophan solution in

hydrochloride acid to formation of tryptophan hydrochloride (100µg.mL-1 in HCl 0.1M). Getting an aliquot 50µg.mL-1 of the sample, add 2.8mL of sulphuric acid 75%, shake in vortex, add 0.1mL cysteine hydrochloride solution 2.5%, shake in vortex again, let in waterbath 45-50°C for 10 minutes, cool at room temperature and add 1mL tryptophan hydrochloride solution, shake again in vortex.

This sequence must be followed rigorously during assay because the formation of the final chromophore depends on the initial formation of the first formed chromophore by complexation with cysteine hydrochloride. After that, read the absorbance in spectrophotometer at 518nm.

5.2 Resorcinol method (Roe et al., 1949)

This reaction follows the same theoretical principles in which there is formation of furfural from hexoses and hydroxy-methyl-furfural (HMF) and from aldopentoses by acid dehydration (Fig. 1). These two products, singly are colorless, however, it is necessary a phenolic compound addition in the medium to develop a colored compound, in this case redness. This technique is firstly mentioned by Roe (1934), with some posterior modifications by Roe et al. (1949), becoming a quick reaction and with stable color. The reaction uses the hydrochloride acid (HCl) for carbohydrates dehydration and the resorcinol is the phenolic compound that reacts with furfural and HMF. This test allows distinguish aldoses from ketoses because the reaction with ketoses is faster and more intense than aldoses. Therefore, the formation of the furfural is easier than HMF formation.

The sensitivity of the method ranges from 10 to 80µg.mL-1 fructose. A standard fructose solution is used to build the calibration curve and to get the equation of the linear regression to quantity samples.

5.2.1 Methodology

Reagents: Resorcinol reagent, 1g resorcinol and 0.25g thiourea in 100mL. This solution must be kept in the dark to keep its stability. Hydrochloride acid is diluted as 5mL HCl and 1mL water.

Getting an aliquot 80µg as maximum of the sample, add 0.5mL resorcinol reagent, shake in vortex, add 3.5mL hydrochloride acid, shake again, let in water bath at 80°C for 10 minutes, after that, read the absorbance in spectrophotometer at 520nm.

Hoping this will be helpful,

Rafik

Thanks prof. Rafik karaman for the idea!