Validation and comparison of analytical methods based on the release of p-nitrophenol to determine lipase activity in milk

Three methods of determining lipase activity were optimized, validated, and compared using skim and whole milk. A chromogenic ester (p-nitrophenyl caprylate) was used in all to quantify the enzyme activity through the release of p-nitrophenol. It was measured colorimetrically (method A) or spectrophotometrically (methods B and C) with a clarifying reagent to render the samples measurable. Methods B and C differed because an inhibiting mixture was used in the latter method to better stop the enzymatic reaction. All the methods were reliable; they were linear in the range of 0 to 300 mU/ml of the enzyme, and the least detection and quantification limits were 9.31 and 31.03 mU/ml of lipase, respectively. Precision, measured as relative standard deviation, was between 1.52 and 4.94%, and mean recoveries ranged between 81 and 90%. Linearity, sensitivity, and accuracy were significantly different among the methods. Methods B and C had better linearity and sensitivity than method A, and the most accurate results were obtained with methods A and B in skim milk. Sensitivity was influenced by the fat content of the samples. On the other hand, the content of lipase did not influence the reliability of any method. Although, all of the methods were useful for routine analysis of quality control of milk, method B was most reliable. Moreover, it would be the method of choice because it was easier and less costly than the other methods.