determination of n-methyl carbamates in a liver determination of n-methyl carbamates in a liver...


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  • Journal of Chemical Technology and Metallurgy, 54, 2, 2019



    Spaska Yaneva1, Iskra Stoykova2, Dancho Danalev3, Lyubov Yotova3


    N-Methyl carbamates are insecticides that inhibit acetylcholinesterase (AChE) causing same symptomatology in acute and chronic exposures. Traces of them could be found in animal tissues, milk, honey, eggs, etc. because of some foods treatment during growth. There is an increasing interest in fast screening methods for detection of differ- ent pollutant groups in the foods. Biosensors are a promising alternative of the existing chromatographic methods such as HPLC, GC, etc. They are fast, easy to use and provide fully acceptable values for the monitoring methods like sensitivity, LOD, LOQ, etc. In order to construct an optical biosensor, AChE is immobilized as a target enzyme for N-methyl carbamates on the surface of new membranes synthesized by a sol-gel technology. The designed bio- sensensor is tested for determination of methomyl, aldicarb, carbofuran, propoxur in liver samples. An appropriate method for sample preparation is also developed. Further, the new method is validated in accordance with document SANTE/11945/2015 covering the required criteria for N-Methyl carbamates determination. The biosensor could detect levels lower than 10 μg/kg which is the maximum residue limit (MRL) for pesticides in foods. Km of 2,2x10-3

    M is calculated for acetylcholine using a 4 month life-time of a biosensor. Keywords: biosensor, pesticide, acetylcholinesterase, N-methyl carbamates, toxic compounds.

    Received 31 October 2018 Accepted 05 December 2018

    Journal of Chemical Technology and Metallurgy, 54, 2, 2019, 362-368

    1Departament of Fundamentals of Chemical Technology, University of Chemical Technology and Metallurgy 8 Kliment Ohridski, Sofia 1756, Bulgaria E-mail: 2Central Laboratory of Veterinary Control and Ecology (CLVCE), Bulgarian Food Safety Agency 5 Iskarsko Shousse str, Sofia, Bulgaria E-mail: 3Department of Biotechnology, University of Chemical Technology and Metallurgy 8 Kliment Ohridski, Sofia 1756, Bulgaria or


    Pesticides and pest management have a significant role in the agriculture industry and the public health field. The residues from pesticides are found in a variety of matrices such as animal tissues, milk, honey, eggs, etc. which is a consequence of their bio-accumulation. The presence of these compounds in the foods constitutes a serious risk to both human and animal health [1]. The Di-

    rective 96/23/EC N-Methyl carbamates, 1996 [2] defines the pesticides as environmental contaminants (group B), and their use is not prohibited. Therefore, some traces of them can be found in foods, but maximum residue levels (MRLs) are set in Regulation 396/2005 for foods of ani- mal and plant origin (REGULATION EC No396/2005) [3]. These documents require each member of the EU to establish annual monitoring program for the control of residues in foods and feed (NMPCR). The specific

  • Spaska Yaneva, Iskra Stoykova, Dancho Danalev, Lyubov Yotova


    requirements for analytical quality control and validation procedures for pesticide residues analysis in foods and feed are described in document SANTE/11945/2015 [4].

    N-methyl carbamates are organic compounds – they are esters of carbamic acid (Fig. 1). They are widely used as insecticides in homes, gardens, and agriculture due to their less toxic effect on the human organism and faster degradation in the environment, compared with those of other groups of pesticides [5]. The mechanism of action of N-methyl carbamate includes reversible carba- moylation of the enzyme acetylcholinesterase (AChE). This reaction allows accumulation of acetylcholine (a neuromediator substance), at parasympathetic neuroef- fector junctions (muscarinic effects), at skeletal muscle myoneural junctions and autonomic ganglia (nicotinic effects), and in the brain (CNS effects). N-methyl car- bamates are hydrolyzed enzymatically in the liver and the degradation products are excreted by the kidneys [6].

    EXPERIMENTAL Analytical standards

    Four certified standards solutions were used: Al- dicarb, of 99,9 % purity, Propoxur, of 99,8 % purity, Carbofuran, of 99,0 % purity and Methomyl, of 100 % purity. All of them were purchased from Dr. Eh- renstorfer (Germany). The working standard solutions were prepared at 6 levels - 0,0; LOQ; 50 %, 100 % and 150 % from the maximum residue level (MRL) for each compound according to their MRL published in Regulation 396/2005 (2005) [3]. All standards were diluted with acetonitrile (a gradient grade) supplied by Supelco (USA).

    Preparation of membranes and AChE immobilization The acetylcholinesterase used in this study was

    isolated from Electrophorus electricus (electric eel) Type VI-S, lyophilized powder, 200 units/mg protein -1,000 units/mg protein. In addition, the following reagents were used - polyamidoamine (PAMAM) dendrimer, ethylenediamine core, generation 4.0 solution Formula: [NH2(CH2)2NH2]:(G = 4); dendri PAMAM(NH2)64 of a molecular mass of 14214.17, methyltriethoxysilane (MTES) and cellulose acetate propionate (САР), all purchased from Sigma-Aldrich. 3g of CAP were homogenized in 40ml of chloroform for 2 h (solution A).

    1ml MTES was mixed with 3mL of ethanol and 1 drop of conc. HCl (solution B).

    The hydrolysis reaction was conducted at а room temperature with vigorous stirring at 25°C for 1 h in a Beher glass. Solution A was combined with solution B and 100µl РAМАМ dendrimer were added. The ob- tained mixture was stirred for 3 h, removed to a petri, and dried at a room temperature.

    The synthesized membranes were activated by treat- ment with 12.5 mL solution of formaldehyde (HCHO) in phosphate buffer (0.1M pH = 7.5) at a temperature of 45oC, and stirring for 4 h in a closed container. The acti- vated membranes were washed by distillated water at the end of the reaction time. The covalently immobilization of enzyme AChE on the membranes was subsequently carried out by adding 1 % enzyme solution to 0.1 M phosphate buffer (pH 5.5). It was spread over the surface of the hybrid membranes and left at 4°C in dark for 8 h.

    The residual activity of the immobilized enzyme was measured for sample analysis. It was then converted to

    Fig. 1. N-methyl carbamates structure formula.

    Traditionally, N-methyl carbamates are analyzed by chromatography techniques such as HPLC, LC-MS or GC [1,7]. AChE-biosensors are a possible alternative suitable for the detection of pesticides. The majority of these biosensors are based on the inhibition reaction of the enzymes acetylcholinesterase (AChE) or butyrylcho- linesterase (BuChE) by the detected substances [8 - 11]. Biosensors are currently a very useful technique for pre- liminary detection of the availability of some substances in environmental monitoring, health care, biological fluids detection, and foods analysis. They represent an alternative method for quick detection of neurotoxins and have been an active research area in the last several years [12 - 16].

    The purpose of the present study is to construct a fiber optic biosensor based on covalent immobilization of AChE on the surface of novel hybrid membranes for the detection of N-methylcarbamate residues in liver samples. In addition, the developed method has been validated in accordance with the official documents in the food control area related to the pesticides monitoring in order to demonstrate the possibility for application of the newly created biosensor in practice.

  • Journal of Chemical Technology and Metallurgy, 54, 2, 2019


    a relative activity (%) and compared with the relative activity of a blank sample. The inhibition percent is calculated as: 100 – relative activity of the sample (%) = inhibition %

    The results were calculated on the ground of a calibration curve obtained with samples spiked with an inhibitor (carbofuran) at 5 levels: LOQ; 0,5 MRL; 1,0 MRL 1,5 MRL and 2,0 MRL. It was built with each series of samples (R2 = 0,9932).

    Enzyme activity determination The enzyme activity of AChE was determined on

    spectrophotometer VWR634-6001, UV-1600PC in ac- cordance with the Worthington methodology [17]. The specific activity of free AChE was defined as:

    Enzymatic assay The method was based upon the disappearance of

    ACh as determined by the ferric-acethydroxamic acid complex. One unit of AChE activity was equivalent to the disappearance of one micromole of ACh per minute at 25°C.

    Sample preparation 5g of the homogenized sample (swine liver) were

    placed into a 50 mL centrifuge tube. Then 5mL of a buffer-substrate solution were added as described in The Worthington Enzyme Manual [17]. The obtained mix- ture was put on a vortex for 2 min, followed by 10 min brake. The sample was then transferred through a filter paper into a glass tube containing 100 mg membranes with an immobilized enzyme. The obtained mixture was vortexed again for 2 min. The solvent was separated from the membranes and AChE activity was determined. The reactivation of the enzyme was performed immediately after its inhibition with 1mM of 2-pyridine aldoxime methyl chloride (2-PAM) water solution for 15 min.

    For quality control, each sample sequence contained a blank sample for the enzyme activity control, and three spiked samples of concentrations corresponding to LOQ; 0,5MRL and MRL levels fro


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