The inhibition of acetylcholinesterase in the presence of selected
organothiophosphates (diazinon, malathion, chlorpyrifos, azinphos-methyl,
phorate) and their oxo-analogues (diazoxon, malaoxon, ...chlorpyrifos-oxon,
azinphos-methyl-oxon, phorate-oxon) was examined. Experimental conditions
were optimized to detect the lowest possible concentrations of these compounds
using AChE test. IC50 values were determined for all the mentioned compounds.
Investigated organothiophosphates have been converted into their
oxo-analogues in the presence of the enzyme myeloperoxidase. Oxidation
products were detected using UPLC 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) and temperature (25°C) were estimated. Oxidation of OPs in the presence
of MPO under optimal conditions was applied as a modification of the method
for detecting OP using AChE test. The detection limits were determined for
the modified method, as well as the efficiency of oxidation and its
application for the analysis of sintetic mixture of OPs. Examined
organothiophosphates have been converted into their oxo-forms using
electrochemically generated halogens. Products have been identified as oxons
using UPLC analysis. It was found that the most efficient oxidation of OPs
was carried out by after 15 minutes of oxidation with electrogenerated
bromine. Detection limits were determined, too. Effects of enzymatic and
electrochemical oxidation on the sensitivity of the AChE test for the
detection of OP were compared. The results showed that the IC50 values were
lower by 100 to 1000 times when the oxidation takes place in the presence of
electrochemically generated halogens and that the limits of detection were
for an order of magnitude lower, compared to the enzymatic oxidation
procedure. The effects of OP and OP oxidized in the presence of MPO were
tested on immobilized AChE. In this case, the method detection limit was for
an order of magnitude lower.
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 potvrđeno pomoću UPLC 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 odgovarajuće 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 određivanje
OP kada su OP oksidovani elektrohemijski generisanim bromom. Upoređeni su
efekti enzimske i elektrohemijske oksidacije OP na osetljivost AChE testa za
detekciju OP. Rezultati su pokazali da su granice detekcije za jedan red
veličine niže kada se oksidacija odvija u prisustvu elektrogenerisanih
halogena u odnosu na enzimsku oksidaciju. Efekat neoksidovanih OP i OP
oksidovanih u prisustvu MPO ispitan je i na imobilizovanoj AChE u protočnom
injekcionom sistemu. U ovom slučaju, granica detekcije metode još je niža, i
to za još jedan red veličine u odnosu na slobodnu acetilholinesterazu.
