Quantification of Malondialdehyde in Colon Tissue

A total of 500 mg of fresh colon tissue was homogenized at 24,000 rpm/min with 1.15 percent KCl. After combining the homogenate with 250 uL of 6M NaOH and incubating at 67 °C for 43 min, the mixture was vortexed with the same amount of acetonitrile. When the samples had been hydrolyzed, they were centrifuged for ten minutes at a speed of 15,000× g. The supernatant (240 μL) was combined with 2,4-dinitrophenylhydrazine (DNPH) (24 μL). For another 10 min, it was kept in the dark. The resultant samples were evaluated by HPLC (Waters Breeze-2, USA) via the ODS2 reverse phase column (Waters Breeze-2, USA). The mobile phase was 38: 62 acetonitrile: 0.2% acetic acid HPLC grade water. MDA in the sample was measured at 310 nm using isocratic HPLC and a UV detector. As per procedure, a standard curve was generated using 20 nmol/mL of MDA standard solution (TCI, Japan) diluted with 1% H₂SO₄ [26 (link),29 ,30 (link)].