Development of a Chromatographic Method for the Determination of Alkaline Phosphatase Activity in Pasteurized Milk

Alkaline phosphatase (ALP) is an important native enzyme of milk which is an established indicator for testing the adequacy of milk pasteurization. In the present study, a HPLC-UV method was standardized for the determination of ALP activity in pasteurized milk in terms of p-nitrophenol concentration liberated from the enzyme substrate di-sodium p-nitrophenyl phosphate. p-Nitrophenol was extracted by clarification, centrifugation, evaporation, and solvent exchange. The analyte was separated by a reversed-phase C18 column using an isocratic mobile phase composed of distilled water and acetonitrile (1:1) in a low-pressure gradient elution mode with a total run time of 12 min. The UV detection was achieved at a wavelength of 319 nm. Values obtained for different validation parameters confirmed that the method was fit to serve the purpose because of good specificity, linearity demonstrated under various experimental conditions (r 2 > 0.9), very low limit of detection (LOD, 0.024 mg/L) and limit of quantitation (LOQ, 0.072 mg/L), accuracy (>86 %), and precision (percent relative standard deviation (% RSD) < 6.152). Validation studies also clearly demonstrated the ability of the HPLC method to detect as low as 0.1 mg/L of p-nitrophenol and 0.0006 % of raw milk. Laboratory evaluation of the HPLC method in comparison with an international reference method (ISO method) showed that the HPLC method has advantages over the ISO method due to lower LOD and LOQ, ability to detect very low levels of raw milk contamination, and comparatively high accuracy and precision.