A new quantitative assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products including whole milk, low fat and skim milks, chocolate milk, and creams. ALP in the test sample hydrolyzes a nonfluorescent substrate, FluorophosR, to a highly fluorescent product. Product formation is monitored continuously during a short incubation period and enzyme activity is calculated from the rate of fluorescence increase. Total test time is 3 min. Reaction rates are linear up to 0.5% raw milk (equivalent to 5 μg phenol/ml/15 min) with a detection limit of 0.006% raw milk. Within and between run precision of the fluorometric method was assessed by repeated analysis of a pasteurized milk sample containing added mixed herd raw milk. The within run (N=10) mean was 190.4 mU/L, standard deviation (SD) 3.2, and a coefficient of variance (CV) of 1.7%. The procedure provides a rapid, sensitive, precise, and easy-to-use ALP assay, applicable to a wide variety of dairy products.

These preliminary results with the use of fluorescence-labeled antibiotics as probes combined with microvolume fluorimetry for the rapid detection of bacterial contamination of platelet components suggest that this is a promising approach. Further studies with additional organisms and alternative conjugates, bacteria, and antibiotics are underway.

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Official methods for the measurement of alkaline phosphatase (ALP) in dairy products use either phenyl phosphate or phenolphthaleln monophosphate as substrate. Quantitation of results requires butanol extraction of the Indophenol (Scharer) or 3-h dialysis of the liberated phenolphthaleln (Rutgers). The Advanced Fluorophos® assay Is based on a self-indicating substrate which, when acted upon by ALP, loses a phosphate radical and becomes a highly fluorescent compound. The rate of fluorophore formation Is monitored for 3 mln In a fluorometer and the enzyme activity In mU/L Is calculated. Eight laboratories participated in a collaborative study to evaluate the Fluorophos® assay for determining ALP activity In whole milk, skim milk, chocolate milk, and cream (half and half). The comparative method was the AOAC quantitative phenyl phosphate method, 16.121-16.122 (14th Ed.). Mixed herd raw milk was added to pasteurized samples at 0.05, 0.1, and 0.2% (v/v). Method performance at 0.1% (v/v) added raw milk as measured by repeatability and reproducibility standard deviations (sr and sR) and relative standard deviations (RSDr and RSDR), respectively, were: whole milk, sr = 21.7%, sR = 34.6%, RSDr = 4.4%, RSDR = 7.0%; skim milk, sr = 19.2%, sR = 31.4%, RSDr = 3.8%, RSDR = 6.2%; chocolate milk, sr = 27.6%, sR = 45.8%, RSDr = 5.3%, RSDR = 8.8%. The method has been adopted official first action by AOAC for determination of alkaline phosphatase in whole milk, skim milk, and chocolate milk.