Development of a HPLC Fluorescence Method for Determining Efficacy of Milk Pasteurization

Alkaline phosphatase (ALP) is an active enzyme present in the raw milk. Due to its unique heat-sensitive properties, it is employed as an indicator to evaluate efficiency of the pasteurization treatment. In the present study, a method comprising of HPLC with fluorescence detector was developed for the quantification of ALP enzyme activity in the pasteurized milk. The method is based on the detection and quantification of 4-methylumbelliferone (4MU) liberated by the action of phosphatase enzyme (in the milk) on the substrate 4-methylumbelliferyl phosphate (4MUP). Extraction and purification of 4MU was done by clarification, centrifugation, and solid phase extraction processes. The target compound separation was achieved with the help of reversed-phase C18 column using gradient elution consisting of a binary mobile phase: HPLC grade water and methanol. The fluorescence detection was accomplished with the excitation and emission wavelengths being 365 and 460 nm, respectively. The method validation experiments clearly showed high specificity, good linearity (r2 > 0.9901), low limits of detection (0.349 mg/L) and quantification (LOQ, 0.432 mg/L), accuracy (> 100%), and precision (%RSD < 4.7652). Comparative evaluation against Lovibond comparator method revealed that the HPLC method has an advantage of detecting very low levels of ALP activity and raw milk contamination. This is the first report of detecting such low levels of ALP in the milk by HPLC-fluorescence detector technique. This new chromatographic method can be considered as a substitute to the existing standard methods that employ fluorometric technique for detecting ALP activity in the pasteurized milk. Our method could certainly enhance the quality assurance standards currently employed in the dairy industry.