Pesticides are used to increase productivity in agriculture and avoid various insects in residential and other public areas. They can remain on food during the process of growing, collecting, distributing, and consuming vegetables and fruits. Moreover, their residues can exist in the air, water, and soil as the result of transferring these molecules. The pesticides have many adverse effects on humans, environment, and other organisms in the ecosystem. Therefore, analysis of pesticides residues in food and environmental samples is an important challenge and closely monitored by most of the legal agencies in Europe and other developed countries. Therefore, identification and determination of pesticide residues is very critical for the human health and wellbeing, as well as for the ecosystem. When pesticides are applied to an area, some of their molecules evaporate and pass to living things through respiration and the other part condenses on the surface of the soil such as snow, rain, and irrigation water. Then, these substances are consumed by plants or water and accumulate in fat tissues [1
As an organic compound, organophosphorus and carbamate pesticides are extensively applied to agricultural areas owing to their great effectiveness, wide spectrum for various species, and low persistence. They are widely used all over the world because they are an attractive alternative to persistent organochlorine pesticides and possess the ability to rapidly degrade under natural conditions sunlight, air, and soil [2
]. The carbamate form is a class of highly effective commercial pesticides and they are preferred worldwide since 1960. They are known as N-substituted carbamic acid esters [3
]. As a carbamate pesticide, propoxur (PRO) was introduced by German chemical manufacturer Bayer in 1975. It is used against insect pests such as chewing and sucking insects, flies, moths, cockroaches, ants, crickets, and mosquitoes [4
]. The toxic properties are based on inhibition of acetyl cholinesterase in the central nervous system, leading to paralysis in insects and mammals [5
]. Fenitrothion (FEN) is one of the most common organophosphorus insecticides frequently used to control insects in crops, such as vegetables, rice, fruits, coffee, soybeans, cotton, and cereals. It is also employed to rein the mosquito vector carrying. FEN is partially water-soluble and therefore may enter the ground and underground water [6
]. Molecular formulas of PRO and FEN pesticides were given in Figure 1
Contamination of surface water and groundwater with hazardous compounds has attracted increasing attention in recent decades all over the world [8
]. According to the European Union (EU) Directive on water quality (98/83/CE) [9
], the maximum admissible concentration (MAC) for pesticides is 0.1 μg L−1
for each individual substance and 0.5 μg L−1
is the maximum allowed for the total concentration of all organophosphorus pesticides. For this reason, development of easy applicable, sensitive, cheap, and correct analysis methods for pesticide molecules is needed as an alternative to the time consuming and expensive chromatographic laboratory techniques currently in use [10
]. A lot of analytical method and pretreatment procedures were studied and published for propoxur and fenitrothion pesticides in various samples such as plant and animal tissues, vegetables, fruits, food grains, different type of waters, and milk samples. These methods are generally based on pretreatment procedures or expensive hybrid instrumental tools [3
Trace analysis of the analytes in complex samples generally requires a pretreatment step to isolate and enrich the target analytes, and to reduce the matrix interferences prior to instrumental determinations [15
]. The most common used pretreatment procedure is solid phase extraction (SPE) based on column or batch type because the application is easy and available for automation. Although many sample preparation techniques have been already used by analytical chemists, solid-phase extraction (SPE) based approaches are mostly preferred in routine analytical process because of its versatility and the possibility of using different materials as adsorbent [16
]. There are a variety of sorbents available for various target ions or molecules. However, separation scientists have been trying to develop a better one every day. An appropriate adsorbent is very crucial for the efficient extraction of the analyte; therefore, the exploration of new adsorbents has received growing attention. Among adsorbent materials, carbon materials have attracted substantial interest due to their high surface area, excellent adsorption capacity, good chemical stability, and low cost [19
]. Magnetic solid-phase extraction (MSPE) is based on magnetic particles as adsorbent that makes the sample preparation procedure simple and fast [21
]. In MSPE, target analytes are adsorbed on the surface of magnetic particles and separated from the sample solution by an external magnetic force easily [16
]. Iron based magnetic particles have significant advantages including primarily price, environmentally safe, high specific surface area, physical and chemical stability, and compatibility for biomedical applications [23
]. In addition to these advantageous properties, their most favorable characteristics include easier separation through an external magnetic field, and the absence of internal diffusion resistance [24
]. Moreover, the required selectivity can be obtained by covering the core with various functional groups for target molecules. These are ideal properties expected from an effective preconcentration and separation method [27
The present study reports a simple, rapid, and convenient procedure based on magnetic solid- phase extraction and HPLC-DAD for the trace determination of propoxur and fenitrothion in environmental water samples. The used magnetic particles as solid-phase sorbent was synthetized and characterized for this study. The sensitivity in this MSPE approach is mainly based on synthesized magnetic particle modified by decanoic acid. The finally, the procedure was applied to water samples successfully.
3. Materials and Methods
3.1. Reagents and Standard Solutions
All reagents used were of analytical grade. Ultra-pure water with a resistivity of 18.2 MΩ was used in all experiments and was provided by an ELGA (Flex III, Lane End, UK) water purification system. Fenitrothion and propoxur pesticide standard were purchased from Sigma-Aldrich (Steinheim, Germany). HPLC grade solvents, methanol, acetonitrile, and isopropyl alcohol were from Merck (Darmstadt, Germany). The stock solutions (200 mg/L) of pesticides were prepared by dissolving each of them in methanol. The working solutions were prepared by appropriate dilution of the stock solutions with double-distilled water. All the standard solutions were stored at 4 °C and brought to ambient temperature just prior to use.
A mixture stock solution containing propoxur and fenitrothion at 100.0 ng mL−1 was prepared in methanol. A series of standard solutions were prepared by mixing an appropriate amount of the stock solution with methanol in a 10 mL volumetric flask. 10 mM of Britton Robinson (BR) buffer (10 mM boric acid, 10 mM glacial acetic acid, and 10 mM phosphoric acid), with 100 mM NaCl was used in all the experiments at different pH values ranging from pH 2.0–10.0.
The chromatographic system used was equipped with a pump model LC20-AD (Shimadzu, Tokyo, Japan), a thermostatic oven, CTO-10 AS (Shimadzu), auto sampler, SIL-20AC (Shimadzu), and a DAD detector model SPD-M20A (Shimadzu). An LC solution software (Shimadzu) was used to transfer data to the computer. A Luna Omega C18 (250 × 4.6 mm, 5 µm) column was used for chromatographic separation. A pH meter with a glass-calomel electrode (Selecta, Spain) was used to measure the pH values. An ultrasonic water bath (Kudos, China) was used for sample preparation.
Previous determinations, all solvents used in the chromatographic system were filtered through a 0.45 µm PDFA membrane filter (HNWP, Millipore, Burlington, MA, USA) using a vacuum pump (Buchi, Switzerland) and degassed for 10 min in an ultrasonic bath (JP Selecta, Barcelona, Spain).
Field-emission scanning electron microscope (Carl Zeiss-Gemini 500, Jena, Germany) was used to examine the morphology of decanoic acid modified magnetic nanoparticles and prove formation of magnetic nanoparticles by EDX analysis. The XRD measurements of decanoic acid modified magnetic nanoparticles was carried out by a Bruker AXS D8 advance X-ray powder diffractometer. FT-IR analysis for decanoic acid modified magnetic nanoparticles was continued by Perkin-Elmer Spectrum 400 FT-IR spectrometer (Waltham, MA, USA). TGA analysis was obtained with a Perkin Elmer-Diamond TGA analyzer. Thermo gravimetric analysis was performed in the range of 50–700 °C, N2 medium, and 10 °C/min−1 temperature rise rate.
3.3. HPLC-PDA Operating Conditions
The mobile phase composition was ACN:water (70:30) at an isocratic mode throughout the analysis. The flow rate was 1.2 mL min−1
. The detector wavelength was operated at 269 and 271 nm for FEN and PRO, respectively. The column temperature was maintained at 30 °C and injection volume was 10 µL for all determinations. The HPLC system was optimized to obtain better signal-to-noise ratio of pesticides in a single chromatographic analysis, the best peak shape, an appropriate run-time, and better peak resolution. The column was flushed with 50% methanol to completely elute the remaining pesticides and the other organic compounds for 60 min after each day’s work. Peaks in the chromatograms were identified by comparison with retention times and UV spectra of standards. The peak area was considered for quantification. The obtained chromatogram after MSPE by using model solutions including PRO and FEN pesticides was presented at four increasing levels in Figure 10
. Absorption spectrums obtained from DAD detector were given as Supplementary 1
3.4. Synthesis of Magnetic Nanoparticles
A well-known synthesis approach in literature was used for obtaining magnetic material [33
]. Briefly, a mixture of 0.745 g of FeCl3
O and 0.383 g of FeSO4
O were solved in 50 mL of 3 M HCl during stirring at a speed of 600 rpm. Then, 100 mL of 50% ethanol was added to the solution and temperature was kept on at 85 °C on a magnetic stirrer. The synthesis reaction was started by dropping 20 mL of ammonia throughout 10 min in inert medium of nitrogen gas. The obtained magnetite Fe3
particles were collected from the black solution by the help of external magnet, washed three times by using 50% MeOH, and dried in the oven at 60 °C for 6 h.
In the second step; 1 g Fe3O4 particles was dispersed in 80% ethyl alcohol solution including 1 mL of ammonia. The mixture was stirred at 600 rpm and 80 °C for 1 h after 1 mL of tetraethyl orthosilicate (TEOS) were added to the solution. Then, 200 mg decanoic acid was solved in 2 mL ethanol and added to the solution on a magnetic stirrer. The mixture was stirred for 6 h. The magnetic particles were removed from the solution by an external magnet and washed five times by 50% ethanol and then allowed to dry.
3.5. The Proposed Method of Magnetic Solid-Phase Extraction
50 mg of the synthesized magnetic material was weighed into a falcon tube and washed two times with 2 mL of ultrapure water. Then, 2 mL pH 7 buffer solution was added on the particles. Then, 20 mL sample solution including pesticides was added to the tubes and the volume of tubes was completed until 50 mL with ultrapure water. For adsorption of target molecules on magnetic particles surface, the tubes were placed on an orbital shaker at 80 rpm for 30 min. At the end of this period, the magnetic solid phase was easily separated by using a Neodymium magnet. After separation of aquatic phase, 400 µL isopropanol (2-propanol) was added to the tubes and they vortexed for 40 s in order to facilitate passing of pesticides molecules to organic phase. Then, the samples were filtered through a 0.45 μm syringe type filter and submitted to HPLC micro vials. After every use, the magnetic particle was washed with 1 mL isopropanol and 1 mL ethanol two times. The solid phase was ready for re-use after drying at the end of this procedure.
3.6. Preparation of Environmental Water Samples
The accuracy and applicability of the proposed method were tested with recovery tests by using various environmental water samples. Samples were prepared by using the methods in literature [37
]. Briefly, natural samples from river, lake, and pond collected in Sivas, Turkey (Kızılırmak River, Tödürge Lake and 4 Eylül pond) were selected for analysis. After collection, the samples were immediately transported to the laboratory and stored in the dark. Prior to the analysis, water samples were filtered through a 0.45-μm PTFE filter and were stored at 4 °C. All samples were analyzed in triplicate using the developed MSPE procedure and HPLC-PDA system. Water samples were also used in this study to calculate the recovery of pesticides at two concentration levels (100 and 300 ng mL−1