Simultaneous enzymatic kinetic determination of pesticides, carbaryl and phoxim, with the aid of chemometrics.

A sensitive and selective enzymatic kinetic method for the simultaneous determination of mixtures of carbaryl and phoxim pesticides was researched and developed. It was based on the inhibitory effect of the pesticides on acetylcholinesterase (AChE), and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as a chromogenic reagent for the thiocholine iodide (TChI) released from the acetylthiocholine iodide (ATChI) substrate. The DTNB-thiocholine reaction was investigated by a spectrophotometric-kinetic approach. The complex rate equation for the formation of the chromogenic product, P, was solved under certain experimental conditions, which enabled the absorbance (A(P), at lambda(max)=412 nm) from the mixtures of the two pesticide inhibitors to be directly related to their concentrations provided the absorbance additivity was followed. The spectra were measured for mixtures of carbaryl and phoxim at different concentrations, and at t=904 s, T=35 degrees C, pH=7.5, c(ATChI)=0.14, and c(AChE)=0.10 mg mL(-1). The detection limits of the enzymatic kinetic spectrophotometric procedures for the determination of the carbaryl and phoxim were 4.7 and 0.59 microg L(-1), respectively. Calibration models for chemometrics methods, such as principal component regression (PCR), partial least squares (PLS) and radial basis function-artificial neural network (RBF-ANN) were constructed and verified with synthetic samples of the mixtures of the two pesticides. The best performing model was based on the RBF-ANN method yielding at approximately 10 ppb analyte concentrations, %RPE(T) (carbaryl=5.2; phoxim=6.5), %Recovery (approx.105%) and %RPE(T) (6.5). Various spiked town-water samples produced recoveries in the range of 98.8-103% for each pesticide.