Identification of Carboxylesterase Metabolites of Residual Malathion in Wheat Kernels Using Semi-Micro Radio Liquid Chromatography

Abstract

Since residual malathion in wheat kernels is shown to be enzymatically decomposed during sample preparation using the Japanese official method for pesticide analysis, the decomposition products were identified by semi-micro radio liquid chromatography (LC). The reaction mixture of the supernatant of wheat kernel homogenate incubated with [14 C] malathion for 0-8 hr was fractionated, and the radioactive decomposition products in the fractions were identified by comparison with the retention time of fifteen reference standards. Twelve of these products occurring in the environment, diethyl fumarate, diethyl maleate, diethyl malate and diethyl succinate, and their hydrolysate and desmethyl malathion dicarboxylic acid were not detected in the reaction mixture, indicating no contribution of organophosphorus hydrolase or methyl transferase. A trace amount of desmethyl malathion was observed, however, this seemed to be non-enzymatically produced. Malathion monocarboxylic acid and malathion dicarboxylic acid were identified as the major products by the LC condition with and without an ion pair reagent, respectively. The half life of malathion decomposition was 2.1 hr, and the reaction time course of these products demonstrated that malathion was decomposed to malathion monocarboxylic acid followed by malathion dicarboxylic acid. These results suggested that residual malathion in wheat kernels was mainly hydrolyzed to malathion carboxylic acids by wheat carboxylesterase during the sample preparation.

Kinetic Analysis for Hydrolysis of Malathion by Carboxylesterase in Wheat Kernels

“…11) However, it could not be elucidated whether this MMC was MMCα, MMCβ or a mixture of the two. The aim of this paper is to characterize wheat kernel CE by identification of the MMC isomers formed from malathion and to investigate the degradability of malathion and each product by the enzyme.…”

“…Dibutyl amine acetate as ion pair reagent for liquid chromatography (LC) was from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). MDC, desmethyl malathion (DMal), and desmethyl malathion dicarboxylic acid, which were synthesized in our previous study, 11) and MMCα and MMCβ, which were newly synthesized, were used as standard reference materials of malathion metabolites. Chemical Synthesis --MMCα: MMCα was synthesized enzymatically as follows; Malathion (2 g) was added to porcine pancreatic lipase solution (20 g/150 ml) in 0.02 M sodium phosphate buffer (pH 7.0) and incubated at 36 • C for 40 hr.…”

“…We previously identified MMC and MDC as malathion metabolites in the supernatant of wheat kernel homogenate, suggesting that there was at least malathion-degradable CE. 11) Malathion is hydrolyzed into both malathion…”

“…9) However, we were previously unable to separately measure MMCα and MMCβ in a mixture of malathion and the supernatant of wheat kernel homogenate, although MMC formation had been observed in the mixture. 11) In this paper, we attempt to identify both MMC isomers as the metabolites of malathion in supernatant of wheat kernel homogenate by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). The degradations of malathion and the identified products were enzymekinetically analyzed to characterize the wheat kernel CE.…”

Characterization and Malathion Degradability of Carboxylesterase in Wheat Kernels

“…The products also suggested that the degradation was due to CE. 9,10) CE hydrolyzes carboxylesters and belongs to the esterase family. 11) 1-Naphthyl acetate and pnitrophenyl acetate are generally recognized as typical substrates of CE.…”