SOP: Determination of Hydroxymethylfurfural

GIFSQ — Global Institute for Food Safety & Quality

Principle

In acid media, hexose sugars break down into hydroxymethylfurfural (HMF), which can polymerize or combine with amino compounds to form brown pigments. HMF reacts with resorcinol in an acid medium to give a reddish-pink colour measured at 540 nm, or with barbituric acid and p-toluidine to give a red colour measured at 550 nm. It can also be estimated from the absorbance of the ether extract at 284 nm (ε = 16,830). Good-quality juices contain little or no HMF. Sulphites interfere with the barbituric-acid method and their interference must be overcome.

Apparatus required

• Blender
• Centrifuge
• Separating funnels
• Filter assembly with Whatman No. 1 and No. 2 papers
• Glass-stoppered test tubes
• Spectrophotometer or colorimeter (readings at 540 nm and 550 nm)

Reagents

a. Ethyl alcohol, 99.9%.

b. 1% resorcinol in HCl.

c. Sodium chloride.

d. Diethyl ether.

e. Filter aid.

f. For the barbituric-acid method: barbituric acid solution (dissolve 500 mg of barbituric acid, previously dried at 105 °C, in warm water and make up to 100 ml in a volumetric flask — prepare fresh); p-toluidine reagent (dissolve 10 g of p-toluidine in 50 ml of isopropanol in a 100-ml volumetric flask, add 10 ml of glacial acetic acid, and make up to volume with isopropanol — prepare fresh); zinc ferrocyanide clarifying solution (Solution A: dissolve 21.9 g of crystallized zinc acetate in water, add 3 ml of glacial acetic acid, and make up to 100 ml with water; Solution B: dissolve 10.6 g of crystallized potassium ferrocyanide in 100 ml of water); 0.1 N iodine solution; 0.5% starch solution; sodium bicarbonate; isopropanol.

Procedure

Resorcinol method

Blend solid samples in a blender. Mix 20 g of the sample thoroughly with water, make up to 100 ml, and centrifuge (for liquid samples, dilute 20 g to 100 ml and centrifuge)

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To 30 ml of the centrifugate add 0.2 g of filter aid and filter through Whatman No. 2 paper under suction

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To 10 ml of the filtrate add 2.5 g of NaCl and extract thrice successively with 20 ml of ether in a separating funnel; combine the ether extracts, taking care not to draw off any emulsified portion

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To the combined extracts add 1 ml of water and evaporate at room temperature in an air draught; make the volume of the residue up to 10 ml; filter using filter aid with Whatman No. 1 paper

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To 3 ml of the filtrate add 3 ml of ethyl alcohol (99.9%) and 3 ml of 1% resorcinol in HCl; mix well, store in the dark for 30 minutes, and measure the reddish-pink colour at 540 nm

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Prepare a blank as in the sample using 3 ml of water in place of sample extract and set the instrument with this blank to 100% transmittance

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From the standard curve (standard solutions containing 0 to 180 mg of hydroxymethylfurfural per ml, developed and measured in the same way — carried as printed in the book), note the concentration and calculate as mg per 100 g

Barbituric acid / p-toluidine method

Take 25 ml of the juice (or concentrate diluted to single-strength juice); adjust the pH to between 7 and 8 with sodium bicarbonate; add a few drops of isopropanol to reduce foaming

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Add 4 ml of starch solution and then iodine solution dropwise until the blue colour persists for 15 seconds (this overcomes sulphite interference)

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Transfer to a 50-ml volumetric flask; add 1 ml each of zinc ferrocyanide solutions A and B with mixing after each addition; make up to volume with water and filter

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Develop the colour in two glass-stoppered test tubes as follows:

• Tube A (blank): 2.0 ml filtrate + 5.0 ml p-toluidine + 1.0 ml distilled water
• Tube B (sample): 2.0 ml filtrate + 5.0 ml p-toluidine + 1.0 ml barbituric acid

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Mix and measure the colour of the sample (B) and the blank (A) at 550 nm within 3 minutes; subtract the blank reading from the sample reading

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Develop the colour with pure HMF exactly as the test sample to get the standard curve, which is linear up to 50 mg

Calculation

Hydroxymethylfurfural = mg per 100 g of sample, read from the standard curve and corrected for the dilutions made.

Precautions

• In the resorcinol method keep the coloured solution in the dark for the full 30 minutes before reading.
• In the barbituric-acid method read the colour within 3 minutes of mixing and always subtract the blank (tube A).
• The book’s standard-curve concentration units (“0 to 180 mg per ml”) are carried as printed; verify against the printed book before quantitative work.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Luh, B. S., Leonard, S. and Marsh, G. L., Food Technol. (Chicago), 12, 347 (1948) — as cited in the book.

International Federation of Fruit Juice Producers, Analyses, No. 12, 1 (1964) — as cited in the book.

Romann, E. and Staub, M., Mitt. Geb. Lebensmittel Unters. Hyg., 52, 44 (1961) — as cited in the book.