SOP: Estimation of Tertiary Butylhydroquinone (TBHQ)
GIFSQ — Global Institute for Food Safety & Quality
Principle
Tertiary butylhydroquinone is extracted from fats, oils or food products with methanol, and estimated colorimetrically with dimethylamine reagent; the colour developed is measured at 500 nm against a calibration curve prepared from standard TBHQ solutions.
Apparatus required
- 50-ml stoppered conical flasks
- 250-ml conical flask
- Soxhlet extraction apparatus
- Wrist-action shaker
- Spectrophotometer
- Rapid-flow, retentive filter paper
Reagents
a. Dimethylamine (DMA) reagent: a 25% solution in water.
b. Standard TBHQ solution: dissolve 0.1 g of TBHQ in methanol and make up to 100 ml with the same solvent; dilute 10 ml of this stock solution to 100 ml with methanol (1 ml = 100 µg).
c. Methanol.
d. n-Butanol.
e. Diethyl ether (for Soxhlet extraction).
Procedure
Preparation of standard curve
Pipette aliquots of the standard solution into 50-ml stoppered conical flasks to give a range of 100 to 1000 µg of TBHQ per aliquot; add methanol to give a total volume of 10 ml; use 10 ml of methanol as blank
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Add 5 ml of DMA reagent to each flask with a pipette, stopper, and shake thoroughly; allow to stand for 30 min
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Add 10 ml of n-butanol to each flask and mix well; measure the colour of the standards at 500 nm, using the blank
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Plot absorbances against concentrations to get the standard curve
Extraction of TBHQ from fats and oils
Weigh 10 to 20 g of oil or melted fat into a 250-ml conical flask
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Add 50 ml of methanol, stopper the flask, and shake for 1 hr in a wrist-action shaker; allow to stand until the two layers separate (1–2 hr)
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If waxy precipitate is observed in the upper methanol layer, filter about one-half of the methanol extract through rapid-flow, retentive filter paper
Extraction of TBHQ from food products
Take a known weight of the food that would give 10 to 20 g of fat — when TBHQ has been added on the basis of percent by weight of the product, take a known weight of the product containing 2 to 4 mg of the antioxidant in the methanol extract
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Extract the product in a Soxhlet apparatus using diethyl ether as solvent for 16 hr; evaporate away most of the ether; remove the last traces by immersing in a water bath held at 30–35 °C
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Dissolve the residue in a known volume of methanol; filter if necessary
Colour development
Take an aliquot of the methanolic extract of the antioxidant (1–10 ml); add enough methanol to make up to 10 ml; use 10 ml of methanol as blank
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Add 5 ml of DMA reagent to each, stopper, shake thoroughly, allow to stand for 30 min, and then add 10 ml of n-butanol; if the solution is not clear, filter through rapid-flow filter paper
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Measure the colour at 500 nm against the blank; read the concentration of TBHQ corresponding to the absorbance from the calibration curve
Calculation
TBHQ % = (C × V₂ × 100) ÷ (V₁ × W × 1,000,000)
Where,
- C = TBHQ in mg per aliquot, as read from the calibration curve
- V₁ = ml of methanol extract taken for analysis
- V₂ = ml of methanol used to extract the fat, oil or food product
- W = weight in g of oil, fat or food product
Note: the printed layout of this formula is partly illegible in the available text (the denominator reads as a garbled string); the expression shown follows the book’s printed numerator (C × V₂ × 100), the printed symbol key (V₁ = aliquot taken, V₂ = solvent used to extract, W = weight), and the printed denominator factor 1,000,000. Consult the printed book for the exact printed form.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.