Fluorimetric-flow injection determination of theophylline based on its inhibitory effect on immobilized alkaline phosphatase

A fluorimetric method for the determination of low levels of theophylline based on the inhibition of the enzyme activity of alkaline phosphatase is proposed. The approach used is a flow-injection configuration where bovine alkaline phosphatase is immobilized on controlled pore glass. The enzymatic reaction is based on the hydrolysis of 4-methylumbelliferone phosphate with fluorimetric monitoring of the 4-methylumbelliferone formed at λex = 365 nm and λem = 445 nm. The presence of theophylline in the samples inhibits the catalytic effect of alkaline phosphatase. The linear determination range was found to be between 0.1 and 200 μmol l−1, with a relative standard deviation < 3.5%. The main common interferent species in serum were tested and they did not produce disturbance at their normal concentration in serum. The applicability of the method was checked by adding the analyte to human serum. The recoveries found were between 93 and 105%.