Journal of Chemical Technology and Metallurgy, 54, 2, 2019
362
DETERMINATION OF N-METHYL CARBAMATES IN A LIVER SAMPLE USING AN OPTICAL BIOSENSOR
Spaska Yaneva1, Iskra Stoykova2, Dancho Danalev3, Lyubov Yotova3
ABSTRACT
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 2018Accepted 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: [email protected] Laboratory of Veterinary Control and Ecology (CLVCE), Bulgarian Food Safety Agency 5 Iskarsko Shousse str, Sofia, Bulgaria E-mail: [email protected] of Biotechnology, University of Chemical Technology and Metallurgy 8 Kliment Ohridski, Sofia 1756, Bulgaria E-mail:[email protected] or [email protected]
INTRODUCTION
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
363
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].
EXPERIMENTALAnalytical 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 immobilizationThe 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
364
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 determinationThe 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 assayThe 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 preparation5g 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 from each of the targeted carbamates for the calibration curve.
Method validationThe validation was performed in accordance
with the recommendations mandated in document SANTE/11945/ [4]. The blank liver samples spiked with N-methyl carbamates at levels corresponding to 0,5; 1 and 1,5 MRLs were used for evaluation of the recovery and precision (repeatability and within-laboratory repro-ducibility). The spiked samples at each concentration level were analyzed in three series, each on a different day, and each in six replicates. The accuracy determined by the average recovery was calculated by comparing the determined concentrations of the spiked samples and their target level. The precision was determined by calculating the coefficient of variation (CV). The limit of quantification (LOQ) of N-methyl carbamates was tested as the minimum concentration of the analyte that could be quantified with an acceptable accuracy and precision. The uncertainty was estimated in accordance with the recommendations of Eurachem/CITAC Guide (2011), [18].
RESULTS AND DISCUSSION
The validation process is carried out in order to confirm that the developed method is applicable for its intended use, and in order to fulfil the aim of this study. Since the result referring to the biosensor is given as a sum of pesticides, the validated method is screening. All validation results obtained for N-methyl carbamates determinations in the spiked liver samples are listed in Table 1. The quantifica-tion of the sum of N-methyl carbamates is obtained using a calibration curve obtained on the ground of samples spiked with carbofuran. The estimated validation parameters of the method are satisfactory. The accuracy of the method expressed as mean recoveries are higher than 70 % for all spiked levels and all tested N-methylcarbamate pesticides. The repeatability of the measurements and the within-labo-ratory reproducibility expressed as coefficients of variation (CV) are lower than 12.7 % and 15.4 %, respectively. The regression curve for all pesticides shows a good linearity with regression coefficients (R2) values higher than 0.95.
The parameters referring to the accuracy and pre-cision obtained for liver samples at the LOQ level of 0.004 μg/g are listed in Table 1. The LOQ values are considerably lower than MRLs as required by document SANTE 11945/2015 [4].
Spaska Yaneva, Iskra Stoykova, Dancho Danalev, Lyubov Yotova
365
The rate of AChE inhibition by carbofuran is the lowest - 33,5 % (Fig. 2).
The results referring to the sum of the pesticides in the sample are estimated on the ground of a calibration curve obtained using samples spiked with carbofuran (Fig. 3). The sample is required to be run by a confir-mation method in case the inhibition percentage result is higher than 30 %. The number of papers reporting
biosensor analyzes conducted with real samples and complying with the requirements in the food control area is limited. There are experiments [19, 20] with food of a plant origin and water because whose fat content is low. That facilitates the sampling. The results from the analysis of carbamates pesticides in milk with a biosensor are presented by Zhang et al. [21]. A sample preparation is not applied because the matrix is in a
Table 1. Validation parameters for a liver sample.
Pesticide Level
[µg/g] Linearity
Recovery [%], (n=18)
Repeatability CV [%], (n=6)
Within laboratory
Reproducibility, CV[%], (n=18)
Measurement uncertainty
[%]
Methomyl
0,010 y = 1788x + 3.9702
R² = 0.9768
83,2 12,73 15,43 12,6
0,020 85,6 8,01 11,35 9,49
0,030 93,1 3,63 4,18 4,87
Aldicarb
0,005 y = 1788x + 3.9702
R² = 0.9768
82,8 10,88 12,01 10,45
0,010 84,1 6,92 12,14 8,62
0,015 92,8 3,81 3,99 4,74
Propoxur
0,025 y = 802.49x + 4.8161
R² = 0.9805
85,0 7,59 7,38 7,48
0,050 85,1 4,28 4,04 4,92
0,075 87,1 4,09 4,24 4,81
Carbofuran
0,005 y = 802.49x + 4.8161
R² = 0.9805
89,2 8,89 8,43 6,98
0,010 88,3 5,13 5,09 5,21
0,015 90,1 3,73 4,14 4,98
Table 2. LOQ for an optical biosensor.
MRL
[µg/g]
LOQ
[µg/g]
Recovery [%],
(n=20)
Within laboratory
Reproducibility, CV[%], (n=20)
Methomyl 0.02 0.004 81,4 13,1
Aldicarb 0.01 0.004 85,3 12,8
Propoxur 0.05 0.004 83,4 12,4
Carbofuran 0.01 0.004 87,9 10,7
Journal of Chemical Technology and Metallurgy, 54, 2, 2019
366
liquid state The resulting LOQ for paraoxone is 0.001 μg/g, while for carbaryl - 0.020 μg/g. This is one order of magnitude higher than those obtained in the present study. Moreover, Zhang et al. use the enzyme just once in their biosensor [21].
The validation process in the present study is per-formed in accordance with the recommendations of document SANCO. It describes the method of validation and the analytical quality control requirements support-ing the validity of the data used in checking the compli-ance with the maximum residue limits, the enforcement actions, or the assessment of the consumer exposure to pesticides in the EU. This document is complementary and integral to the requirements of ISO/IEC 17025 (2005) [22]. The validation parameters obtained for a liver matrix demonstrate that the developed analytical method meets the method performance acceptability criteria (mean recoveries in the range 70 % - 120 %,
precision with CV < 20 %, LOQ < MRL).In contrast to other biosensor tests described in the
literature that reach low recoveries (mostly below 70 %) [23] the recovery data obtained in this study cor-respond to the required minimum of 70%. Schulze et al. [20] report paraoxon-ethyl yield of 101% in peach purée and 106 % in apple purée [20]. The biosensor constructed by them is incubated directly in the food, which is not applicable in the present work due to the type of samples being tested, namely the liver. Samples of an animal origin cause additional interfering effects when compared to those of a vegetable origin because of the fat content.
It is found in current work that the higher the con-centration of the pesticides in the sample is, the better are the repeatability results. At the second validation level, all repeatability results are below 8%. This is less than half of the requirement value. It means that the biosensor works
Fig. 2. An ihibition percentage of AChE at MRL level.
Fig. 3. A calibration curve for the optical biosensor constructed.
Spaska Yaneva, Iskra Stoykova, Dancho Danalev, Lyubov Yotova
367
very precisely and the results are with minimal deviations under recurring conditions. The lowest value of CV for repeatability is obtained with methomyl at a validation level of 3.00 % - 3.63 %, i.e. 5 times below the criterion.
It is concluded on the ground of the repeatability and reproducibility data that the biosensor precision in-creases with the increase of the pesticide concentration. This is a prerequisite to consider a minimal possibility of obtaining false negative results. Moreover, because of the high specificity of the reaction, practically all of AChE inhibitors can be detected even at the low levels demanded by the food control. This is extremely impor-tant for screening methods as it is far more dangerous to have a false negative result than a false positive. There are also stringent measures - no more than 5 % of the samples (2002/657/EC, 2002) [24].
CONCLUSIONSThe newly developed biosensor offers many advantages
over the chromatographic methods. It works directly with real samples, so any specific preliminary sample preparation is not necessary. The method requires a simple sample preparation procedure. In addition, the biosensor corresponds to all requirements of EU documents related to the parameters of the food testing methods such as sensitivity, detection limit, linearity, within-laboratory reproducibility. It has sensibility similar to that of the chromatographic methods that are currently used for determination of N-methyl carbamates. Finally, the biosensor has a shorter response time, a much lower cost and easier maintenance. It is very suitable for a preliminary control providing a positive or negative response prior to proceeding to more expensive and time consuming laboratory analysis. The increase of the pesticide concentration in the sample increases also the precision. The results obtained in the current study demonstrate without any doubt that biosensors can be introduced for food control as an alternative screening method for pesticide analysis.
AcknowledgementsThe article is in memoriam of Prof. Dr. Lyubov Yo-
tova. This study is particularly financed by the project DN 07/21 with National Scientific Fund of Bulgaria.
Ethical approval: This work describes the construc-tion and the application of a biosensor for determination of pesticides in food matrices only so no animals or humans are objects of this study.
REFERENCES
1. M. Le Doux, Analytical methods applied to the determination of pesticide residues in foods of animal origin. A review of the past two decades, J. Chromatogr. A, 1218, 2011, 1021-1036.
2. Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC, OJ L 125, 23.05.1996.
3. Regulation (EC) N: 396/2005 of the European Par-liament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC.
4. SANTE/11945/2015: Guidance document on ana-lytical quality control and validation procedures for pesticide residues analysis in food and feed.
5. P.R. Jäger, N. Costin, K. Heinz, Carbamates and Carbamoyl Chlorides, Ullmann’s Encyclopedia of Industrial Chemistry, Weinheim, Wiley-VCH, 2005, doi:10.1002/14356007.a05_051.
6. Micromedex I, Registry of Toxic Effects of Chemi-cal Substances: NIOSH, 1991.
7. J. Zhang, H.K. Lee, Application of liquid-phase microextraction and on-column derivatization com-bined with gas chromatography–mass spectrometry to the determination of carbamate pesticides, J. Chromatog. A, 1117, 2011, 31-37.
8. M. Ahmed, J.B. Rocha, C.M. Mazzanti, A.L. Morsch, D. Cargnelutti, M. Corrêa, V. Loro, V.M. Morsch, M.R. Malathion, Carbofuran and Paraquat Inhibit Bungarus Sindanus (Krait) Venom Acetyl-cholinesterase and Human Serum Butyrylcholinest-erase in vitro, Ecotoxicology, 16, 2007, 363-369.
9. S. Drvesh, K.V. Darvesh, R.S. McDonald, D. Mataija, R. Walsh, S. Mothana, O. Lockridge, E. Martin, Carbamates with Differential Mechanism of Inhibition Toward Acetylcholinesterase and Butyrylcholinesterase, J. Med. Chem., 51, 2008, 4200-4212.
10. R. Sinha, M. Ganesana, S. Andreescu, L. Stanciu, AChE Biosensor based on Zinc Oxide Sol-gel for the Detection of Pesticides, Analytica Chimica Acta, 661, 2010, 195-199.
Journal of Chemical Technology and Metallurgy, 54, 2, 2019
368
11. L. Yotova, N. Medhat, Optical biosensor with multienzyme systems immobilized onto hybrid membrane for pesticides determination, Int. J. Bio-automation, 15, 4, 2012, 267-276.
12. T.M.A. Gronewold, U. Schlecht, E. Quandt, Analy-sis of Proteolytic Degradation of a Crude Protein Mixture using a Surface Acoustic Wave Sensor, Biosensors and Bioelectronics, 22, 2007, 2360-2365.
13. X.S. Guo, Y.Q. Chen, X.L. Yang, L.R. Wang, Novel Shear-horizontal Surface Acoustic Wave based Im-munosensors using SiO2 Waveguiding Layers and Flow Injection Analysis, Proc. of the International Conference of IEEE Engineering in Medicine and Biology Society, 2, 2005, 1921-1924.
14. Z. Hanane, J.L. Cisneros, L.B. De Naranjo-Rod-riguez, K.R. Temsamani, J.L. Marty, Alumina Sol-gel/Sonogel-carbon Electrode based on Acetyl-cholinesterase for Detection of Organophosphorus Pesticides, Talanta, 77, 2008, 217-221.
15. C.B. Jacobs, M.J. Peairs, B.J. Venton, Review: Carbon Nanotube based Electrochemical Sensors for Biomolecules, Analytica Chimica Acta, 662, 2010, 105-127.
16. V. Vamvakaki, N.A. Chaniotakis, Pesticide Detec-tion with a Liposome-based Nano-biosensor, Bio-sensors and Bioelectronics, 22, 2007, 2848-2853.
17. N.J. Freehold, Worthington Enzyme Manual, 1972, Worthington Biochemical Corporation.
18. EURACHEM/CITAC, 2011, Guide CG 4 Quantify-ing Uncertainty in Analytical Measurement.
19. V. Pedrosa, J. Caetano, S. Machado, M. Bertotti, Determination of Parathion and Carbaryl Pesticides in Water and Food Samples Using a Self Assembled Monolayer/Acetylcholinesterase Electrochemical Biosensor Sensors, 8, 8, 2008, 4600-4610.
20. H. Schulze, S. Vorlova, F. Villatte, T. Bachmann, R.D. Schmid, Design of Acetylcholinesterases for Biosensor Applications, Biosensors and Bioelec-tronics, 18, 2003, 201-209.
21. Y. Zhang, S.B. Muench, H. Schulze, R. Perz, B. Yang, R.D. Schmid, T.T. Bachman, Disposable biosensor test for organophosphate and carbamate insecticides in milk, J. Agric. Food Chem., 53, 13, 2005, 5110-5115.
22. ISO/IEC 17025, 2005, General requirements for the competence of testing and calibration laboratories.
23. H. Schulze, M. Anastassiades, S. Vorlova, R.D. Schmid, T.T. Bachmann, Development, validation, and application of an acetylcholinesterase-biosensor test for the direct detection of insecticide residues in infant food, Biosensors and Bioelectronics, 17, 2002, 1095-1105.
24. 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results.
