Resorcinol monophosphate ester as a new substrate for fluorometric detection of alkaline phosphatase activity in milk products

Alkaline phosphatase (ALP) is an indicator of sterilization efficacy of milk products and its activity monitoring is essential for ensuring food safety and controlling product quality. Here, a novel fluorometric method for the determination of ALP activity based on resorcinol monophosphate ester (RME) as a new substrate was proposed to verify pasteurization of milk products. RME was cheap and was simply and novelly synthesized by a two-step method using resorcinol (RC) as the raw material. The mechanism is, after dephosphorylation, RME was transformed to RC, which subsequently react with dopamine (DA) to form a strong fluorescent product, azamonardine. It has a fluorescence emission wavelength at 461 nm under the optimal excitation at 418 nm. The results show that under the optimum conditions, there is a good linear relationship between ΔF (the variation of fluorescence intensity) and ALP activity over 0–5000 mU/L. High sensitivity was achieved (detection limit of 17.34 mU/L) because of high quantum yield of the products and catalytic activity of ALP and the recovery rate is 96.8–106.1%. Compared with national and international standard methods, the detection procedures are simple, just adding RME and DA solution to milk with buffer solution. Our method was successfully applied for ALP activity detection in milk products. Moreover, the catalyst of ALP and the reaction of RME and dopamine are pH dependent, and thus the reaction time could be kept identical by terminating the reaction through adding acids. With high sensitivity, high selectivity, simple operation, and wide linear range, our assay will find more practical applications in food analysis.