2.4. TBARS Assay

The thiobarbituric acid reactive substance (TBARS) assay is an analytical method that quantifies rancidity caused by secondary oxidation compounds in high-lipid food systems. This method gives a measure of the aldehydes present in a food product. After a period of time, unstable primary oxidation compounds (hydroperoxides) break down and aldehydes or ketones are formed. Malondialdehyde (MDA) reacts with thiobarbituric acid (TBA) and trichloroacetic acid (TCA) to form a pinkish-red colored complex. The absorbance of this oxidized chromogen, MDA, is measured at 531 nm using a spectrophotometer. The AOCS Cd 19-90 method [17] was used for the TBARS assay, where 5 g of peanut butter bite sample was dissolved in 15 ml of distilled water and homogenized thoroughly. To prepare TCA solution, 20 g of trichloroacetic acid (TCA) was dissolved in 100 ml distilled water and mixed thoroughly. This solution was kept in a closed glass bottle and stored under a fume hood. To prepare the TCA-TBA reagent, 0.6 g of thiobarbituric acid (TBA) was dissolved in 120 ml of TCA solution and mixed thoroughly. This solution was kept in a cool place in a closed glass bottle. 2 ml of the TCA-TBA reagent was added to 1 ml of peanut butter homogenate and 50 μl of butylated hydroxyanisole was added immediately to avoid further oxidation during the sample analysis. The solution was heated for 15 minutes in a boiling water bath and then cooled for 10 minutes in ice water. The mixture was centrifuged at 2000 G for 10 min (Fisher Scientific Model 225 centrifuge). Absorbance was measured using a spectrophotometer at 531 nm wavelength. A standard curve was developed to calculate the concentration of MDA from absorbance values.