Tc. Rodrigues et al., Flow-injection spectrophotometric determination of paraoxon by its inhibitory effect on the enzyme acetylcholinesterase, ANAL SCI, 17(5), 2001, pp. 629-633
A spectrophotometric enzymatic flow injection (FI) system for the determina
tion of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method w
as based on the determination of the acetic acid formed by the enzymatic re
action of the acetylcholinesterase, immobilized on glass beads, with the su
bstrate acetylcholine. The acetic acid formed permeates through a PTFE memb
rane and is received by a solution (pH 7.0) containing the acid-base indica
tor 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 concen
tration 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) consid
ering 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 quantitat
ive 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.