A highly sensitive kinetic spectrophotometric determination for alkaline phosphatase (ALP) using 5,10,15,20-tetrakis (4-phosphonooxyphenyl)porphine (approximately Delta epsilon approximate to 3.0 x 10(5) I mol(-1) cm(-1)) was studied. With a measuring time of 30 min, the determination range was 2.0 x 10(-12)-8.0 X 10(-9) mol l(-1) and the detection limit (3s) was 1.0 x 10(-12) mol l(-1). The detection limit became lower on increasing the measuring time, For example, the detection limit was up to 10(-15) mol l(-1) for 12 h. This method was applied to the assay of ALP in human serum. Using 0.50 ml of sample without any pre-treatment, a good correlation was obtained with the p-nitrophenyl phosphate method, and the relative standard deviation was 4.7%. The method was also applied to the enzyme immunoassay of alpha-fetoprotein in human serum and provided a good correlation with the 4-methylumbelliferone derivative (r = 0.999) with approximately half the relative standard deviations (1.3-2.7%) obtained with a commercial kit. The proposed method requires very small sample volumes, is highly sensitive, highly accurate, and shows good stability of the reagent; it will be applied to various assays using ALP as label.
