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5-Hydroxymethylfurfural (HMF) is separated using HPLC and reversed phases. This substance is measured with a UV detector.
HPLC analysis specifically detects 5-hydroxymethylfurfural.

A photometric method, B-590.59.111 Hydroxymethylfurfural - photometrisch, using barbituric acid and o-toluidine detects all aldehydes present in the sample. This method serves as an alternative for laboratories without HPLC.

5-Hydroxymethylfurfural (HMF) is detected in a color reaction with barbituric acid and o-toluidine. However, this detects all of the aldehydes present in the sample. The determination by HPLC specifically detects the HMF. As with other aldehydes, HMF reacts with barbituric acid and p-toluidine to create a reddish compound. HMF reacts with any sulfurous acid that is present, as do other aldehydes; in this case, it is, therefore, undetectable without prior treatment. If more than 10 mg/l of free sulfurous acid are present, this will bind to acetaldehyde. Subsequently, the determination can be performed. The presence of excess acetaldehyde does not interfere with the analysis.

D-Sorbit is present in pomaceous fruit and stone fruit.

D-Sorbitol is oxidized to D-fructose by nicotinamide adenine dinucleotide (NAD) in the presence of the enzyme sorbitol dehydrogenase (SDH), forming reduced nicotinamide adenine dinucleotide (NADH).

D-Sorbitol + NAD^{+ \(\xrightarrow{SDH}\)}D-G-6-P fructose + NADH + H⁺

The reaction equilibrium heavily favors the reactants NAD+ and D-sorbitol. It shifts toward D-fructose, when the NADH formed in this reaction reacts with iodine nitrotetrazolium chloride (INT) in a subsequent reaction. Formazan is created in the presence of diaphorase, which is an irreversible reaction.

NADH + INT + H⁺ \(\xrightarrow{diaphorase}\) NAD⁺ + Formazan

The maximum absorbance of the formazan is measured at 492 nm.