A novel method was proposed for the determination of five benzimidazoles (oxfendazole, mebendazole, flubendazole, albendazole, and fenbendazole) using magnetic graphene (G-Fe3O4). G-Fe3O4 was synthesized via in situ chemical coprecipitation. The properties of G-Fe3O4 were characterized by various instrumental methods. G-Fe3O4 exhibited a great adsorption ability and good stability towards analytes. Various experimental parameters that might affect the extraction efficiency such as the amount of G-Fe3O4, extraction solvent, extraction time, and desorption conditions were evaluated. Under the optimized conditions, a method based on G-Fe3O4 magnetic solid-phase extraction coupled with high-performance liquid chromatography was developed. A good linear response was observed in the concentration range of 0.100–100
Benzimidazoles (BMZs) are broad-spectrum anthelmintics and have been used in animal husbandry for the prevention and control of a wide variety of gastrointestinal nematodes in aquaculture, agriculture, and veterinary practices [
Sample preparation affects nearly all subsequent assay steps and is critical to the unequivocal identification, confirmation, and quantification of analytes [
In order to enhance the selectivity and extraction of MSPE, magnetic particles are usually subjected to surface functionalization with appropriate recognition molecules. Fe3O4 nanoparticles (NPS) are the most popular particles due to their low cost and toxicity. Fe3O4 NPS have been modified and functionalized with several different materials. Specifically, various research groups have functionalized the NPS with C8 [
In this present study, magnetic graphene was synthesized by in situ chemical precipitation and was used as an adsorbent for the first time. Moreover, a novel analytical methodology based on G-Fe3O4 MSPE coupled to HPLC was developed for the trace analysis of five BMZs, namely, oxfendazole, mebendazole, flubendazole, albendazole, and fenbendazole in chicken, chicken blood, and chicken liver samples.
Graphite powder (50 mesh) was purchased from Boaixin Chemical Reagents (Baoding, China). Potassium permanganate (KMnO4), concentrated sulphuric acid (H2SO4), hydrogen peroxide (H2O2), hydrochloric acid (HCl), nitric acid (HNO3), sodium nitrate (NaNO3), all phosphate compounds, and all ammonium compounds were purchased from Tianjin Tianda Chemical Reagent Co. (Tianjin, China). Oxfendazole (OXF), mebendazole (MEB), flubendazole (FLU), albendazole (ALB), and fenbendazole (FEN) were purchased from Sigma (Sigma-Aldrich, USA). Stock standard solutions of BMZs in dimethyl sulfoxide/methanol (1 : 9, v/v) (400 mg/L) were prepared and were subsequently diluted. HPLC-grade acetonitrile was obtained from Tedia (Fairfield, OH, USA). Ultrapure water used throughout the experiments was obtained from a Milli-Q gradient A10 system (Millipore, UK). All solutions were filtered through a 0.45-
X-ray diffractometry (XRD) was carried out using a Rigaku diffractometer. The size and morphology of the G-Fe3O4 were determined by scanning electron microscopy (SEM), which was conducted using a 4300 SEM instrument (HITACHI, Japan); Transmission electron microscopy (TEM) was carried out using a Philips Tecnai 10 TEM instrument (Philips, Netherlands). Fourier transform infrared (FT-IR) spectroscopy was carried out on a Nicolet Avatar 330. The magnetic properties were characterized using a Squid-based magnetometer from Quantum Design (San Diego, CA). Centrifugation during sample preparation was performed in a TGL-20LM-B centrifuge equipped with angular rotor (12 × 2.0 mL) (Hunan Star Science Instruments Co. Ltd., China). A QL-901 Vortex (Kylin-bell Lab Instruments Co., Ltd., China) was used for preparing the samples. An Agilent 1200 series LC system equipped with a quaternary pump, autosampler, and VWD ultraviolet detector controlled by Chemstation software was used in all analyses.
GO was prepared from natural graphite powder, using a previously reported procedure [
G-Fe3O4 was synthesized by the in situ chemical coprecipitation of Fe3+ and Fe2+ in an alkaline solution in the presence of GO. A solution of NaOH/diethylene glycol (DEG) (10 mg/mL) was prepared by adding 2.0 g sodium hydroxide to 200 mL of DEG; the mixture was refluxed for 1 h at 120°C under a N2 atmosphere and then cooled to 70°C. The magnetic composite was prepared by suspending 0.4 g GO in 250 mL of DEG, the mixture was ultrasonicated for 2 h and added to 1.6 g ferric chloride with stirring for 1 h at room temperature. Then, before rapidly injecting 67 mL of the NaOH/DEG solution, it was heated at 220°C within 30 min. After the product was cooled to room temperature, the precipitate was isolated using a magnetic field, and the supernatant was separated from the precipitate by decantation. The impurities in G-Fe3O4 were removed by washing with water. G-Fe3O4 was then washed with absolute alcohol until the green yellow colour disappeared. Subsequently, the composite was dried at 80°C for 24 h under vacuum.
The chicks were fed using 5 mg/Kg of BMZs in the corn for seven days. Then, they were slaughtered three days later. The thoroughly homogenized chicken, chicken blood, and chicken liver samples were prepared as follows: 5.0 g of the samples, 20 mL of ethyl acetate, 0.30 mL of a 25 g/100 mL of KOH solution, and 0.50 mL of a 1 g/100 mL butylated hydroxytoluene solution were mixed in a 50 mL Eppendorf vial. After the solution was sonicated for 5 min, 0.50 g Na2SO4 was added and was subjected to centrifugation at 16000 rpm. The resulting clear solution was placed in a 100 mL pear-shaped bottle. Next, the same solution was used to wash the homogenizer and the solution was thoroughly vortexed at room temperature for 2 min, sonicated, and subjected to centrifugation again. Subsequently, the clear solution was added to the aforementioned pear-shaped bottle and dried at 40°C by a rotary evaporator. The residue was immediately dissolved in 10 mL of acetonitrile via ultrasonication, and 10 mL n-hexane was added. The acetonitrile solution was collected and dried via distillation under reduced pressure. Then, the residue was dissolved in 15.0 mL of water for the G-Fe3O4 sorptive extraction. The concentrations of BMZs in the spiked sample solutions were 0.80 ng/g and 8.0 ng/g.
The MSPE procedure consisted of extraction, desorption, and HPLC analysis. First, 15.0 mg of G-Fe3O4 was rinsed with acetone and water and dispersed in 15.0 mL of the BMZ water sample solution. Secondly, the mixture was shaken for 30 min to extract the analytes. Subsequently, G-Fe3O4 was isolated from the solution using a magnet placed at the bottom of the beaker; then, the supernatant was poured off. In order to completely remove the residual solution with a pipette, the particles were moved with a magnet, which was placed on the outside of the bottle wall. The isolated particles were then vortexed with 1.0 mL of acetic acid and methanol (1 : 99, v/v) for 25 min to desorb the analytes. Afterwards, the magnet was placed on the bottom of the bottle and the desorption solution was collected with a micropipette and was subsequently dried with N2, to redissolve with 400
All chromatographic separation was performed on a Diamosil C18 (250 × 4.6 mm i.d., 5
X-ray diffraction (XRD) measurements were employed to investigate the structure of the synthesized samples; the graphite and G-Fe3O4 patterns are shown in Figure
(a) X-ray diffraction (XRD) of graphite, Fe3O4, and G-Fe3O4, (b) FT-IR spectra of G-Fe3O4, (c) thermogravimetric analysis (TGA) of G-Fe3O4, and (d) VSM magnetization curves of G-Fe3O4.
The FT-IR spectra illustrated in Figure
The thermal stability of G-Fe3O4 was investigated by thermogravimetric analysis (TGA). As shown in Figure
G-Fe3O4 should possess sufficient magnetic properties to allow for rapid separation under a magnetic field. The VSM magnetization curves of G-Fe3O4 at 25°C are shown in Figure
The TEM and SEM images of the G-Fe3O4 composite are shown in Figure
(a) TEM and (b) SEM images of G-Fe3O4 composites.
The extract was based on
In order to determine the optimum amount of adsorbent (G-Fe3O4) for the extraction of BMZs (OXF, MEB, FLU, ALB, and FEN), the dosages of G-Fe3O4 were varied from 6.0 to 21.0 mg. As shown in Figure
Effect of experimental conditions on the extraction efficiency. (a) Effect of the amount of sorbent and (b) effect of extraction time.
The analytes should be completely desorbed from the G-Fe3O4 particles prior to HPLC-UV analysis. The desorption of BMZs required that the
Effects of experimental conditions on the desorption efficiency. (a) Effects of desorption solvent, A: acetonitrile; B: methanol; C: acetic acid/acetonitrile (1 : 99, v/v); D: acetic acid/methanol (1 : 99, v/v); (b) desorption time; and (c) volume of desorption solvent.
In this study, adsorption capacity was defined as the maximum amount of BMZs extracted by G-Fe3O4. The G-Fe3O4 sorbent was characterized in terms of capacity, which was directly related to the amount of graphene. The extraction capacity of the G-Fe3O4 sorbent was determined by exposing the sorbent to water solutions containing increasing amounts of BMZs (0.050–15.0 mg/L) for 30 min. The results are shown in Figure
(a) Adsorption capacity of G-Fe3O4 and (b) reusability of G-Fe3O4 in the extraction of BMZs.
In order to investigate the reusability of the G-Fe3O4 sorbent, it was washed twice with 5 mL of acetic acid and methanol (1 : 99, v/v) and 5 mL of acetone before it was reused in subsequent MSPE. The experimental results (Figure
Under the optimized conditions, some analytical performance parameters of the method, including linear range (LR), correlation coefficient
A series of working solutions containing OXF, MEB, FLU, ALB, and FEN at concentrations ranging from 0.100 to 100
Analytical performance and results for HPLC-UV determination of five BMZs using magnetic graphene.
Compounds | Equation of linearity |
|
Range | LOD |
LOQ |
RSD |
---|---|---|---|---|---|---|
( |
(ng/L) | (ng/L) | (%) |
|||
OXF |
|
0.9966 | 0.100–100 | 19.4 | 58.7 | 3.4 |
MEB |
|
0.9992 | 0.100–100 | 28.3 | 84.6 | 5.7 |
FLU |
|
0.9998 | 0.100–100 | 27.4 | 82.8 | 7.6 |
ALB |
|
0.9988 | 0.100–100 | 32.3 | 97.4 | 5.4 |
FEN |
|
0.9986 | 0.100–100 | 17.2 | 52.2 | 4.9 |
The MSPE-HPLC method developed in this study was used to analyze several food samples, including chicken, chicken blood, and chicken liver samples. The MSPE showed maximal elimination of the matrix interferences and enhancement of the sensitivity. The amounts of OXF, MEB, FLU, and FEN in these samples ranged within 13.0–20.2, 1.62–4.64, 1.94–6.42, and 0.292–1.04 ng/g, respectively. ALB was also detected in some samples; the results are shown in Table
Analysis of BMZs in food samples using the magnetic solid-phase extraction coupled to HPLC
Samples | Analytes | Original amount (ng/g) | RSD (%) | Spiked concentration (ng/g) | |||
---|---|---|---|---|---|---|---|
0.80 ng/g | 8.0 ng/g | ||||||
Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | ||||
Chicken | OXF | 13.0 | 3.9 | 84.1 | 4.2 | 94.8 | 3.4 |
MEB | 3.00 | 7.2 | 83.0 | 4.9 | 105 | 3.4 | |
FLU | 4.56 | 6.2 | 84.8 | 5.9 | 105 | 5.1 | |
ALB | 0.450 | 6.3 | 89.6 | 5.8 | 107 | 4.8 | |
FEN | 1.04 | 8.7 | 84.7 | 7.9 | 92.4 | 6.4 | |
|
|||||||
Chicken blood | OXF | 17.5 | 3.7 | 93.0 | 3.9 | 93.6 | 4.2 |
MEB | 1.62 | 5.1 | 95.7 | 3.0 | 101 | 2.9 | |
FLU | 1.94 | 6.4 | 102 | 4.7 | 102 | 4.1 | |
ALB | N.Q. | — | 95.7 | 4.3 | 112 | 3.5 | |
FEN | 0.292 | 8.9 | 87.2 | 6.0 | 94.6 | 4.5 | |
|
|||||||
Chicken liver | OXF | 20.2 | 3.5 | 85.9 | 4.2 | 90.9 | 3.5 |
MEB | 4.64 | 5.6 | 88.7 | 5.1 | 115 | 4.3 | |
FLU | 6.42 | 4.3 | 91.8 | 3.4 | 108 | 2.7 | |
ALB | 0.342 | 5.7 | 97.8 | 3.9 | 112 | 3.4 | |
FEN | 1.01 | 6.9 | 95.0 | 4.0 | 97.3 | 3.6 |
Chromatograms of BMZs in chicken, chicken blood, and chicken liver samples. (a) the standard solution at 1.0 mg/L, (b) a sample solution extracted by G-Fe3O4 MSPE, and (c) 0.8 ng/g of the spiked sample solution extracted by G-Fe3O4 MSPE. The peaks corresponded to the following BMZs: 1. OXF, 2. MEB, 3. FLU, 4. ALB, and 5. FEN.
In the present study, G-Fe3O4 was facilely synthesized by in situ chemical coprecipitation and it exhibited a great adsorption ability and good stability in the MSPE of BMZs. The proposed method for the determination of BMZs in chicken, chicken blood, and chicken liver samples was established by combining G-Fe3O4 as an effective adsorbent with HPLC-UV. The LODs were in the range of 17.2–32.3 ng/L, and the recoveries of the method ranged between 83.0% and 115%; the RSDs were less than 7.9%. Furthermore, G-Fe3O4 could be reused at least 30 times without a significant loss in the sorption capacity or magnetism. Moreover, the G-Fe3O4 exhibited a remarkable preconcentration ability for five BMZs, and satisfactory repeatability and recoveries were obtained. The use of G-Fe3O4 was also faster and less expensive than other previously reported methods. The developed method serves as a simple and highly efficient extraction and preconcentration technique for BMZs in chicken, chicken blood, and chicken liver samples. MSPE based on the G-Fe3O4 can also be used for the enrichment of other trace organic pollutants in food samples.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors would like to thank the National Natural Science Foundation of China (no. 21505115) and the Science and Technology Foundation of the Guizhou Provincial Science and Technology Department (nos. LH20147406 and LH20167034).