Flow-injection spectrophotometric determination of paraoxon by its inhibitory effect on the enzyme acetylcholinesterase

A spectrophotometric enzymatic flow injection (FI) system for the determination of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method was based on the determination of the acetic acid formed by the enzymatic reaction of the acetylcholinesterase, immobilized on glass beads, with the substrate acetylcholine. The acetic acid formed permeates through a PTFE membrane and is received by a solution (pH 7.0) containing the acid-base indicator Bromocresol Purple (B. C. P.), leading to a pH change and therefore to a color change. The variation of the absorbance of the solution is detected spectrophotometrically at 400 nm. The determination of paraoxon is related to its inhibitory action on the enzyme. Therefore the analytical signal is the difference between the signal that corresponds to the free and the one that corresponds to the inhibited enzyme, considering a Bred acetylcholine concentration. The correlation between the peak height and paraoxon concentration at a given acetylcholine concentration is linear in the range from 5.0 x 10(-7) mol L(-1) to 5.0 x 10(-5) mol L(-1) (r = 0.998) of paraoxon, with a relative estimated standard deviation (R.S.D.) of +/-1.7% (n = 10) considering a solution containing 5.0 x 10(-6) mol L-l of paraoxon and a solution containing 5.0 x 10(-3) mol L-l of acetylcholine. Therefore, the quantitative limit detection is about 2.5 x 10(-7) mol L(-1) of paraoxon (3 sigma). A 1,1 ' -trimethylene-bis(4-formylpyridinium bromide)dioxime (TMB-4) solution was used to reactivate the enzyme.