Primena oksidacije organo-tiofosfatnih pesticida u metodama za njihovu detekciju na bazi inhibicije acetilholinesteraze

Abstract

Ispitana je inhibicija slobodne acetilholinesteraze (AChE) odabranim organotiofosfatima (OP) (diazinon, malation, hlorpirifos, azinfos-metil, forat) i njihovim oksoanalozima (diazokson, malaokson, hlorpirifos-okson, azinfos-metil-okson, forat-okson), pri čemu su optimizovani uslovi za detekciju najniže koncentracije tih jedinjenja primenom AChE testa. Određene su IC50 vrednosti za sva navedena jedinjenja. U cilju povećanja osetljivosti AChE testa, ispitivani organo-tiofosfati prevedeni su u okso-analoge u prisustvu enzima mijeloperoksidaze (MPO). To je potvreno pomoćuUPLC i GC/MS analize. Nastali oksidacioni proizvodi stabilni su najmanje 1h. Maksimalne koncentracije oksona dobijaju se kada se organo-tiofosfati inkubiraju sa 100 nM MPO u prisustvu H2O2 koncentracije 50 μM, pri pH 6, na temperaturi 25 °C u toku 10 minuta. Oksidacija OP u prisustvu MPO pod navedenim uslovima primenjena je u modifikaciji metode za detekciju OP na bazi inhibicije AChE. Određena je granica detekcije modifikovane metode, kao i efikasnost oksidacije i njena primena na smešu OP. Gore navedeni organo-tiofosfati prevedeni su u odgovarajue oksone i pomoću elektrohemijski generisanih halogena. Proizvodi koji nastaju identifikovani su kao oksoni pomoću UPLC analize. Utvrđeno je da je najefikasnija oksidacija elektrogenerisanim bromom u toku 15 minuta. Određena je i granica detekcije AChE testa za odreivanje OP kada su OP oksidovani elektrohemijski generisanim bromom...

The inhibition of acetylcholinesterase in the presence of selected organothiophosphates (diazinon, malathion, chlorpyrifos, azinphos-methyl, phorate) andtheir oxo-analogues (diazoxon, malaoxon, chlorpyrifos-oxon, azinphos-methyl-oxon,phorate-oxon) was examined. Experimental conditions were optimized to detect the lowestposible concentrations of these compounds using AChE test. IC50 values were determinedfor all the mentioned compounds.Investigated organothiophosphates have been converted into their oxo-analogues inthe presence of the enzyme myeloperoxidase. Oxidation products were detected usingUPLC and GC/MS analysis. It was found that the products are stable within minimum 1h.In order to optimize oxidation procedure and achieve maximum concentrations of oxons,the optimal concentrations of H2O2 (50 μM) and MPO (100 nM) were determined. Also,the optimal incubation time of OPs and MPO (10 min), as well as the optimal pH (6) andtemperature (25 °C) were estimated. Oxidation of OPs in the presence of MPO underoptimal conditions was applied as a modification of the method for detecting OP usingAChE test. The detection limits were determined for the modified method, as well as theefficiency of oxidation and its application for the analysi of sintetic mixture of OPs.Examined organothiophosphates have been converted into their oxo-forms usingelectrochemically generated halogens. Products have been identified as oxons using UPLCanalysis. It was found that the the most efficient oxidation of OPs was carried out by after15 minutes of oxidation with electrogenerated bromine. Detection limits were determined,too.