The reaction mixture was made to a volume of 4 mL with the addition of 0.7 mL of double distilled water and heated at 95°C for 1 hour in a water bath. After heating, 1 mL of double distilled water and 5 mL of a mixture of n-butanol and pyridine (15 : 1 v/v) were added and the mixture was shaken vigorously on a vortex mixer for 5 min. This mixture was then centrifuged at 3000 rpm for 7 min. After centrifugation the upper organic layer was separated and the amount of MDA formed in this layer was measured at 532 nm using an ultra violet/visible spectrophotometer (Systronics, India). Appropriate controls were used at different steps during this estimation (extinction coefficient of MDA is 1.45 × 10−5/min/cm).