Determination of Histamine and Putrescine in Milk by UV-Vis Spectrophotometry Combined with Double Enzyme Colorimetry

A method for determination of histamine and putrescine in milk by UV-Vis spectrophotometry based on diamine oxidase and horseradish peroxidase double enzyme colorimetry was established. 2% (mass fraction) lead nitrate standard solution of 10 mL was added into the milk sample of 10 mL, and mixed well. Then 0.1% (mass fraction) trichloroacetic acid standard solution of 20 mL was added. Atter mixing and centrifuging, the supernatant was taken and adjusted its acidity to pH 7.2. 1.0 g·L^-1 diamine oxidase standard solution of 100 μL was added into sample solution of 100 μL in water bath at 50℃ for 15 min. After the reaction, 1.0 g·L^-1 horseradish peroxidase standard solution of 50 μL, 0.5 g·L^-1 tetramethylbenzidine standard solution of 200 μL and 2 mol·L^-1 hydrochloric acid solution of 50 μL were added successively. The absorbance of the system was measured by UV-Vis spectrophotometer. It was showed that the color of sample solution underwent colorless, and then changed from blue to yellow. Linear relationships between concentrations of histamine and putrescine and their absorbances were kept in the ranges of 2.5-150 μmol·L^-1 and 5-200 μmol·L^-1, with detction limits (3S/N) of 0.652 2 μmol·L^-1 and 1.134 1 μmol·L^-1, respectively. Recovery test was made on blank milk sample, giving results in the ranges of 80.5%-88.3% for histamine and 89.5%-92.3% for putrescine, and RSDs (n=5) of the determined values were in the ranges of 2.5%-4.4% for histamine and 4.4%-5.1% for putrescine.