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Chunqiang Ruan Xiang Zhao Chenglan Liu Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, P. R. China Received October 17, 2014 Revised May 8, 2015 Accepted June 1, 2015
Research Article
Determination of diflubenzuron and chlorbenzuron in fruits by combining acetonitrile-based extraction with dispersive liquid–liquid microextraction followed by high-performance liquid chromatography In this study, a simple and low-organic-solvent-consuming method combining an acetonitrile-partitioning extraction procedure followed by “quick, easy, cheap, effective, rugged and safe” cleanup with ionic-liquid-based dispersive liquid–liquid microextraction and high-performance liquid chromatography with diode array detection was developed for the determination of diflubenzuron and chlorbenzuron in grapes and pears. Ionicliquid-based dispersive liquid–liquid microextraction was performed using the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate as the extractive solvent and acetonitrile extract as the dispersive solvent. The main factors influencing the efficiency of the dispersive liquid–liquid microextraction were evaluated, including the extractive solvent type and volume and the dispersive solvent volume. The validation parameters indicated the suitability of the method for routine analyses of benzoylurea insecticides in a large number of samples. The relative recoveries at three spiked levels ranged between 98.6 and 109.3% with relative standard deviations of less than 5.2%. The limit of detection was 0.005 mg/kg for the two insecticides. The proposed method was successfully used for the rapid determination of diflubenzuron and chlorbenzuron residues in real fruit samples. Keywords: Benzoylurea insecticides / Dispersive liquid-liquid microextraction / Fruits / High-performance liquid chromatography / Ionic liquids DOI 10.1002/jssc.201401162
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Benzoylurea insecticides (BUs) are powerful insect growth regulators that can control the development, reproduction and metamorphosis of target insects by interfering with the normal activity of their endocrine system [1]. Since their introduction, BUs have been widely used to prevent and treat insects in modern agriculture because of their attractive Correspondence: Associate professor Chenglan Liu, Key Laboratory of Natural Pesticide and Chemical Biology, South China Agricultural University, No.483, Wushan Road, Tianhe District, Guangzhou 510642, P. R. China E-mail: [email protected] Fax: +86-21-85280293.
Abbreviations: BU, benzoylurea insecticide; IL-DLLME, ionicliquid-based dispersive liquid–liquid microextraction; DAD, diode array detector; UVD, ultraviolet detector; DSPE, dispersive solid-phase extraction; PSA, primary secondary amine; MRL, maximum residue limit; GPC, gel permeation chromatography; ME-SWATLD, maceration extraction with self-weighted alternating trilinear decomposition; PLE, pressurized liquid extraction. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
properties, such as insecticidal activities, tremendous selectivity, rapid degradation and low mammalian toxicity [2]. However, the presence of pesticide residues in foods resulting from the widespread use of BUs could lead to chronic exposure and long-term toxicity effects. Consequently, the monitoring of benzoylurea insecticide residues in agricultural products has become essential to ensure food safety and to prevent the bioaccumulation of pesticide residues throughout the food chain. Because of the complexity of the matrices in environmental samples, sample preparation is necessary and often plays a vital role in the overall analytical scheme. LLE and SPE are the most common and widely used sample preparation methods for residue analysis [3, 4]. Recently, two types of microextraction, LPME and SPME, which are based on the miniaturization of conventional LLE and SPE, respectively, have been widely used for sample pretreatment [5,6]. In 2006, Rezaee et al. developed a novel liquid–liquid microextraction method, dispersive liquid–liquid microextraction (DLLME) [3]. In DLLME, a water-immiscible extractant and a watermiscible disperser solvent are mixed and rapidly injected into an aqueous sample to form a cloudy solution. This procedure www.jss-journal.com
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significantly increases the interface area and results in good extraction performance. Many studies have demonstrated excellent extraction efficiencies for organic and inorganic analytes using this microextraction technique [7–12]. In the conventional DLLME method, organic solvents that have a density higher than that of water are employed as extractants, and they can be separated and deposited after centrifugation. However, only a few solvents, which are typically highly toxic chlorinated solvents, can be used with this technique because of its specific requirements [13]. The application of ionic liquids (ILs), which contain organic cations and anions that are liquid at room temperature, as extraction solvents in DLLME avoids the primary problems associated with the use of hazardous solvents because of their low volatilities (negligible vapor pressures), chemical and thermal stabilities, and high solubility [14]. In recent years, ILs have been increasingly applied as the extraction solvent, replacing the volatile solvent in DLLME for pesticide residue analysis [15–17]. The DLLME method is widely recognized due to its simplicity, low cost and high enrichment, thus making it available to most analytical laboratories. Unfortunately, the lack of purification for samples with more complex matrices, such as fruits and vegetables, has caused this method to be limited to those with simpler matrices, specifically water and a few fruit juices. The QuEChERS extraction procedure is the most common technique for multi-residue pesticide analysis in foods, particularly fruits and vegetables [18]. The major drawback of this technique is its poor enrichment factor; the technique has higher detection limits compared with other techniques. Researchers have proposed a new method that consists of DLLME preconcentration after QuEChERS extraction [19–21]. Coupling these techniques takes advantage of the benefits of both methods while reducing some of the drawbacks. To the best of our knowledge, there are only a few reports on the extraction and enrichment of pesticide residues in agricultural products using the QuEChERSDLLME method [22–26]. Several sample preparation techniques, such as SPE [4,27], DLLME [28,29] and SPME [30], have been used for the extraction and cleanup of benzoylurea insecticides in fruits, vegetables, fruit juices and water. However, there are no published studies about the pretreatment of diflubenzuron and chlorbenzuron in fruits using the QuEChERS-DLLME method. The present study reports the development of a reliable and sensitive method for the simultaneous determination of two benzoylurea insecticides (diflubenzuron and chlorbenzuron) in grapes and pears. The method is based on liquid–liquid partitioning with acetonitrile and salts (QuEChERS) combined with an ionic-liquid-based dispersive liquid– liquid microextraction (IL-DLLME) followed by HPLC with diode array detection (DAD).
2 Materials and methods 2.1 Chemicals and reagents Diflubenzuron and chlorbenzuron standard solutions (100 mg/L, purity > 99%) were obtained from the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Agro-Environmental Protection Institution, Ministry of Agriculture (Tianjin, China). 1-Hexyl-3-methylimidazolium hexafluorophosphate ([HMIM][PF6 ], ࣙ99.0% purity), 1-butyl3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ], ࣙ99.0% purity), 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM][PF6 ], ࣙ99.0% purity) and 1-octyl-3methylimidazolium tetrafluoroborate ([OMIM][BF4 ], ࣙ99.0% purity) were purchased from Shanghai Cheng Jie Chemical, China. HPLC-grade solvents methanol and acetonitrile, primary secondary amine (PSA) and graphitized carbon black (GCB) were purchased from Shanghai ANPEL Scientific Instrument, China. An ultrapure water purification system with UV+UF optional accessories (UNIQUE-R20, Research, China) was used throughout this study. A 0.22 m cellulose membrane filter was used for filtration of the fruit samples. Stock solutions of diflubenzuron and chlorbenzuron were prepared at 100 mg/L in methanol and were stored in amber glass vials at –20⬚C. Serial dilutions of the standard working solutions were prepared in methanol.
2.2 Instruments and equipment An Agilent 1260 HPLC system (Agilent Technologies, Germany) was used for chromatographic analysis and consisted of a binary pump, automatic sample injector, degasser, and a diode array detector. The software was OpenLAB CDS ChemStation Edition Drivers Revision C.01.05. All separations were performed on a KR100-10 C18 column (5 m, 150 mm × 4.6 mm, Kromasil). The mobile phase consisted of the following gradient program: the initial mobile phase was 60:40 water/MeOH for 2 min, then a 22 min linear gradient from 60:40 to 15:85 water/MeOH, then a linear gradient from 15 to 10% water in 4 min, and the initial eluent composition was restored over a 4 min gradient and was maintained for 4 min. The injection volume was 20.0 L, and the flow rate was 1.0 mL/min. The detection wavelength was 258 nm. An ultrasonic cleaner (SCQ-2201B, Shanghai, China) and a model 5804R centrifuge (Eppendorf, Hamburg, Germany) were used in the experiment.
2.3 Samples Pear and grape samples were purchased from local supermarkets in Guangzhou, China. Samples were crushed homogeneously and stored at –20⬚C until analysis. For recovery determination, 10.0 g of the samples was spiked with different volumes of standard solutions and stored at room temperature for 2 h before analysis.
2.4 Extraction procedure Ten grams of homogenized fruit sample was weighed in a 50 mL PTFE centrifuge tube and extracted with 20 mL MeCN for 10 min in an ultrasonic cleaner. After the addition of 3 g of anhydrous sodium sulfate and 1.5 g of sodium chloride, www.jss-journal.com
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the mixture was vortexed for 1 min and then centrifuged at 3000 rpm for 5 min. A 10 mL sample of the upper MeCN layer was concentrated using a rotary evaporator at 40⬚C to dryness and was reconstituted with 1.6 mL of MeCN and transferred for clean-up by mixing with 100 mg of PSA and 150 mg of MgSO4 . The supernatant was collected after vortexing for 1 min and was centrifuged for 5 min at 3000 rpm. The sample was then filtered through a 0.22 m cellulose membrane filter before the DLLME step.
2.5 DLLME procedure A 1.1 mL aliquot of the acetonitrile extract solution that acted as the dispersive solvent for the DLLME was transferred into a centrifuge tube containing 90 L of ionic liquid [HMIM][PF6] (as the extraction solvent) and was then vortexed for 1 min. The mixture was rapidly injected into a 10 mL centrifuge tube containing 5 mL of 10% NaCl solution. This ternary component mixture was vortexed for 90 s to form an emulsified solution followed by centrifugation for 5 min at 3000 rpm. Finally, the upper aqueous phase was removed using a Pasteur pipette. The volume of the sedimented IL-phase (90 ± 3 L) was re-dissolved with 50 L of MeOH and was vortexed for 60 s. The solution was transferred to a sample vial, and 20 L was injected for HPLC analysis.
3 Results and discussion 3.1 Sample extraction and cleanup by QuEChERS A modified version of the QuEChERS method for sample preparation of fruits was used [31]. The procedure involves initial single-phase extraction of a 10 g sample with 20 mL of acetonitrile, followed by liquid–liquid partitioning by the addition of 3 g of anhydrous MgSO4 plus 1.5 g of NaCl. Normally, MeCN is a good extraction solvent for matrices with low fat content and does not significantly dissolve the matrix interfering compounds [32]. However, to facilitate the DLLME procedure, dispersive solid-phase extraction (DSPE), in which PSA was selected as the absorbent, was adopted to further purify the MeCN extract. In this procedure, different amounts of PSA (50, 100 and 200 mg) were tested. The recoveries obtained from the different PSA amounts were satisfactory (higher than 90%). However, the purification effect of 100 mg PSA was better than that of 50 mg PSA (Supporting Information Fig. S1). Ultimately, 100 mg of PSA was selected as the optimum quantity.
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[16, 33, 34]. When [BMIM][PF6 ] and [OMIM][BF4 ] were used as the extractants, there was limited or no sedimented phase formation, and progression to the next step was very difficult. However, when [HMIM][PF6 ] was used as the extraction solution, satisfactory extraction recoveries and enrichment factors were obtained (Fig. 1A and B). In the next step, the effect of the volume of [HMIM][PF6 ] on the extraction recoveries was investigated in the range of 80–120 L in 10 L intervals. As shown in Fig. 2A, the recoveries for diflubenzuron and chlorbenzuron varied from 72.9–90.2%. The highest recoveries for the two insecticides were obtained when 90 L of [HMIM][PF6 ] was used. Consequently, 90 L of [HMIM][PF6 ] was used as the optimum quantity. In our study, the solvent MeCN was used as the extraction solvent and then as the disperser solvent in the IL-DLLME step. To study the effect of the volume of dispersive solvent on the extraction efficiency, different volumes of QuEChERS extract, from 800–1200 L in 100 L increments, mixed with 90 L of extractive solvent were added to 5 mL of ultrapure water. As indicated by the results shown in Fig. 2B, higher extraction recoveries for the two insecticides were obtained when 1.1 mL of MeCN was used. Thus, 1.1 mL of the QuEChERS extract was chosen as the optimum volume for the dispersive solvent. IL-DLLME experiments were conducted using different amounts of NaCl (0, 5, 10 and 15%, m/v) under the same experimental conditions to test the salting-out effect. From the results shown in Fig. 3, increases of extraction recoveries for the two insecticides from 0–10% NaCl were observed, and the highest extraction recoveries were obtained at 10% w/v of NaCl. Therefore, 10% NaCl was added during the IL-DLLME procedure.
3.3 Matrix effects Different matrix components can be co-extracted with compounds of interest, causing matrix effects. Matrix components have an observable effect on the detection of target analytes by either enhancing or weakening their signal intensities. The matrix effects were evaluated by comparing the relative detector responses with the ratio of the calibration curves slopes obtained from the matrix-matched standard solutions and direct injection of MeOH solutions [35]. Matrix effects can be classified depending on the decrease/increase in the percentage of the slope: between ±20 and 0% represents a mild effect, between ±20 and ±50% is considered a medium effect and higher than ±50% is considered a strong effect of signal suppression or enhancement. The results did not show any matrix effects for the two pesticides (the ratios were close to 1.0 under all conditions).
3.2 Optimization of IL-DLLME To replace the highly toxic halogenated hydrocarbons used in previously reported DLLME methods, four ILs, namely, [HMIM][PF6 ], [BMIM][PF6 ], [OMIM][PF6 ] and [OMIM][BF4 ], were tested according to the methods in previous studies C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 Method validation Under the optimal conditions, the LODs, LOQs and linearity were investigated to validate the proposed method. Ten grams www.jss-journal.com
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Figure 1. Effect of different types of extractant on the recoveries (A) and enrichment factor (B) of BUs. Extraction conditions: 5.0 mL of water, 1.0 mL of MeCN, 0.1 mL of ILs and 0% NaCl.
Figure 2. Effect of the volume of [HMIM][PF6 ] (A) and MeCN (B) on the recoveries of BUs. Extraction conditions: (A) 5.0 mL of water, 1 mL of MeCN and 0% NaCl; (B) 5.0 mL of water, 90 L of [HMIM][PF6 ] and 0% NaCl. Table 1. Linear ranges, LODs, LOQs, average recoveries and repeatability (%RSD) obtained with the QuEChERS-IL-DLLME method in spiked fruits, analyzed by HPLC-DAD (n = 5 at each level) Pesticides
Fruits
Linearity equation
Linearity (g/mL)
r
MRLa ) (mg/kg) China
Diflubenzuron Chlorbenzuron
Grape Pear Grape Pear
y = 1959.4x – 1.1066 y = 1596.4x – 5.0874 y = 2260.9x – 4.1439 y = 1794.7x – 4.1542
0.01-0.5 0.01-0.5 0.01-0.5 0.01-0.5
0.9998 0.9996 0.9997 0.9995
c)
— 1 — —
LOD (mg/kg)
LOQ (mg/kg)
Japan 0.05 1 — —
0.005 0.005 0.005 0.005
0.01 0.01 0.01 0.01
Recovery (%) ± RSD (%) Level 1b)) 0.01
Level 2 0.05
Level 3 0.2
102.9 ± 0.5 101.3 ± 0.6 99.5 ± 1.0 102.9 ± 1.4
98.6 ± 1.0 107.2 ± 3.3 99.7 ± 1.93.4 100.9 ± 5.2
109.3 ± 0.6 106.8 ± 0.6 106.6 ± 3.4 101.3 ± 1.0
a) The data of MRLs from China Pesticide information Network. Available at . b) The unit of the concentration was mg/kg. c) No MRL be established at present.
of fruit samples spiked at five increasing concentration levels were subjected to the previously optimized methods described in Section 2.4 to obtain the calibration curves. The results are summarized in Table 1. The calibration graphs showed linearity for the concentration ranges of diflubenzuron and chlorbenzuron. Good correlation coefficients equal to or greater than 0.9995 were achieved for diflubenzuron and chlorbenzuron. The LODs were determined by injecting serial sample extraction solutions based on the S/N ratio of 3:1. The lowest fortified level was used as the LOQ. The LOD and LOQ for the two insecticides were 0.005 and 0.01 mg/kg, respectively. The LODs and LOQs of the present method were C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
below the MRLs established in China and Japan. The MRL of diflubenzuron was 0.05 mg/kg for grape and 1 mg/kg for pear in Japan. In China, the MRL of diflubenzuron for pear was 1 mg/kg. For chlorbenzuron, there are no MRLs for grape and pear in Japan and China (Table 1). Recovery assays were performed with the blank fruits samples (grape and pear) spiked at three concentration levels of 0.01, 0.05 and 0.2 mg/kg for diflubenzuron and chlorbenzuron. Each level was performed in five replicates as described in Section 2. The results are listed in Table 1. The relative recoveries for diflubenzuron and chlorbenzuron ranged from 98.6–109.3% at the three spiking levels, and the RSDs varied www.jss-journal.com
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Figure 3. Effect of the NaCl percentage on the recoveries of BUs. Extraction conditions: 5.0 mL of water, 90 L of [HMIM][PF6 ] and 1.0 mL of MeCN.
Figure 4. Typical chromatograms of BUs in spiked and blank fruit samples prepared by QuEChERS-IL-DLLME. (1) Diflubenzuron; (2) chlorbenzuron.
from 0.5–5.2%. Representative chromatograms of the BUs in the spiked and blank fruit samples from the supermarkets in Guangdong Province, China, are illustrated in Fig. 4.
traces of diflubenzuron or chlorbenzuron residues (data not shown). However, a more thorough and long-term investigation of fruit-derived BUs residues is necessary to ensure consumer safety.
3.5 Analysis of fruit samples The validated analytical method was used to monitor diflubenzuron and chlorbenzuron residues in ten fruit samples (five grapes and five pears) purchased from the local supermarkets in Guangdong Province, China. No samples showed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4 Comparison of QuEChERS-IL-DLLME with other methods Comparison of the presented QuEChERS-IL-DLLME method with several published methods demonstrated that the www.jss-journal.com
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Table 2. Comparison of the proposed methods and some other methods for the determination of benzoylureas residues in fruits samples
Method
Sample mass (g)
Organic solvent
LOD (g/kg)
Recovery (%)
Ref.
GPC-HPLC-UVD ME-SWATLD-HPLC-DAD SPE-HPLC-DAD PLE-LC-MS/MS QuEChERS-IL-DLLME-HPLC
50 50 25 5 10
Acetone (100 mL) +CH2Cl2 (50 mL) Methanol (50 mL) Acetonitrile (50mL) +(Petroleum ether+ Acetone) (65 mL) Ethyl acetate (25 mL) +Methanol (5 mL) Acetonitrile (20 mL)+ [HMIM][PF6] (90 L)
10–30 17–440 20–50 0.7–2 5
71–90 91–116 80–120 58–97 98–109
[36] [37] [38] [39] Proposed method
proposed method exhibited satisfactory extraction performance in terms of the relative recoveries and precisions (Table 2) [36–39]. At the same time, the LODs with QuEChERS-IL-DLLME were better than most of the other methods (data shown in Table 2). This can be explained by the fact that high enrichment factors could be obtained by DLLME. A high enrichment factor can also be obtained by increasing the quantity of analyzed samples and extraction solvent in conventional methods. However, this will also increase the consumption of organic solvents and the cost of the overall experiment. Additionally, the proposed method has the advantage of lower consumption of organic solvents (approximately 20 mL of organic solvent, data shown in Table 2). According to the comparison, the QuEChERS-ILDLLME method was concluded to be rapid, effective and environmentally friendly.
[3] Rezaee, M., Assadi, Y., Hosseini, M. R. M., Aghaee, E., Ahmadi, F., Berijani, S., J. Chromatogr. A 2006, 1116, 1– 9. [4] Miliadis, G. E., Tsiropoulos, N. G., Aplada-Sarlis, P. G., J. Chromatogr. A 1999, 835, 113–120. [5] Paillakis, E., Kalogerakis, N., TrAC, Trends Anal. Chem. 2003, 22, 565–574. [6] Zambonin, C. G., Cilenti, A., Palmisano, F., J. Chromatogr. A 2002, 967, 255–260. [7] Aguirre, M. A., Selva, E. J., Hidalgo, M., Canals, A., Talanta 2015, 131, 348–353. [8] Clavijo, S., del Rosario Brunetto, M. Cerda` V., J. Sep. Sci. 2014, 37, 974–981. [9] Han, D., Yan, H. Y., Row, K. H., J. Sep. Sci. 2011, 34, 1184– 1189. [10] Leng, G., Lui, G. B., Chen, Y., Yin, H., Dan, D. Z., J. Sep. Sci. 2012, 35, 2796–2804. [11] Li, S. Q., Gao, P., Zhang, J. H., Li, Y. B., Peng, B., Gao, H. X., Zhou, W. F., J. Sep. Sci. 2012, 35, 3389–3395.
5 Conclusions
[12] Zhao, E. C., Zhao, W. T., Han, L. J., Jiang, S. R., Zhou, Z. Q., J. Chromatogr. A 2007, 1175, 137–140.
In this study, a novel analytical method, combining QuEChERS extraction with IL-DLLME procedures followed by HPLC–DAD, was successfully developed for the extraction, cleanup and preconcentration of diflubenzuron and chlorbenzuron in fruit samples. The combination of QuEChERS with DLLME provides an inexpensive sample pretreatment that ensures a high enrichment factor and low detection limits. The satisfactory extraction efficiency allows the determination of BUs below the maximum content allowed by China, even using DAD detection. QuEChERS combined with DLLME provides a useful method for the analysis of BUs in fruits for routine laboratory food quality analysis and safety control.
[13] Zgloa-Grzeskowiak, A., Grzeskowiak, T., TrAC, Trends Anal. Chem. 2011, 30, 1382–1399.
The authors have declared no conflict of interest.
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Chunqiang Ruan Xiang Zhao Chenglan Liu Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, P. R. China Received October 17, 2014 Revised May 8, 2015 Accepted June 1, 2015
Research Article
Determination of diflubenzuron and chlorbenzuron in fruits by combining acetonitrile-based extraction with dispersive liquid–liquid microextraction followed by high-performance liquid chromatography In this study, a simple and low-organic-solvent-consuming method combining an acetonitrile-partitioning extraction procedure followed by “quick, easy, cheap, effective, rugged and safe” cleanup with ionic-liquid-based dispersive liquid–liquid microextraction and high-performance liquid chromatography with diode array detection was developed for the determination of diflubenzuron and chlorbenzuron in grapes and pears. Ionicliquid-based dispersive liquid–liquid microextraction was performed using the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate as the extractive solvent and acetonitrile extract as the dispersive solvent. The main factors influencing the efficiency of the dispersive liquid–liquid microextraction were evaluated, including the extractive solvent type and volume and the dispersive solvent volume. The validation parameters indicated the suitability of the method for routine analyses of benzoylurea insecticides in a large number of samples. The relative recoveries at three spiked levels ranged between 98.6 and 109.3% with relative standard deviations of less than 5.2%. The limit of detection was 0.005 mg/kg for the two insecticides. The proposed method was successfully used for the rapid determination of diflubenzuron and chlorbenzuron residues in real fruit samples. Keywords: Benzoylurea insecticides / Dispersive liquid-liquid microextraction / Fruits / High-performance liquid chromatography / Ionic liquids DOI 10.1002/jssc.201401162
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Benzoylurea insecticides (BUs) are powerful insect growth regulators that can control the development, reproduction and metamorphosis of target insects by interfering with the normal activity of their endocrine system [1]. Since their introduction, BUs have been widely used to prevent and treat insects in modern agriculture because of their attractive Correspondence: Associate professor Chenglan Liu, Key Laboratory of Natural Pesticide and Chemical Biology, South China Agricultural University, No.483, Wushan Road, Tianhe District, Guangzhou 510642, P. R. China E-mail: [email protected] Fax: +86-21-85280293.
Abbreviations: BU, benzoylurea insecticide; IL-DLLME, ionicliquid-based dispersive liquid–liquid microextraction; DAD, diode array detector; UVD, ultraviolet detector; DSPE, dispersive solid-phase extraction; PSA, primary secondary amine; MRL, maximum residue limit; GPC, gel permeation chromatography; ME-SWATLD, maceration extraction with self-weighted alternating trilinear decomposition; PLE, pressurized liquid extraction. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
properties, such as insecticidal activities, tremendous selectivity, rapid degradation and low mammalian toxicity [2]. However, the presence of pesticide residues in foods resulting from the widespread use of BUs could lead to chronic exposure and long-term toxicity effects. Consequently, the monitoring of benzoylurea insecticide residues in agricultural products has become essential to ensure food safety and to prevent the bioaccumulation of pesticide residues throughout the food chain. Because of the complexity of the matrices in environmental samples, sample preparation is necessary and often plays a vital role in the overall analytical scheme. LLE and SPE are the most common and widely used sample preparation methods for residue analysis [3, 4]. Recently, two types of microextraction, LPME and SPME, which are based on the miniaturization of conventional LLE and SPE, respectively, have been widely used for sample pretreatment [5,6]. In 2006, Rezaee et al. developed a novel liquid–liquid microextraction method, dispersive liquid–liquid microextraction (DLLME) [3]. In DLLME, a water-immiscible extractant and a watermiscible disperser solvent are mixed and rapidly injected into an aqueous sample to form a cloudy solution. This procedure www.jss-journal.com
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significantly increases the interface area and results in good extraction performance. Many studies have demonstrated excellent extraction efficiencies for organic and inorganic analytes using this microextraction technique [7–12]. In the conventional DLLME method, organic solvents that have a density higher than that of water are employed as extractants, and they can be separated and deposited after centrifugation. However, only a few solvents, which are typically highly toxic chlorinated solvents, can be used with this technique because of its specific requirements [13]. The application of ionic liquids (ILs), which contain organic cations and anions that are liquid at room temperature, as extraction solvents in DLLME avoids the primary problems associated with the use of hazardous solvents because of their low volatilities (negligible vapor pressures), chemical and thermal stabilities, and high solubility [14]. In recent years, ILs have been increasingly applied as the extraction solvent, replacing the volatile solvent in DLLME for pesticide residue analysis [15–17]. The DLLME method is widely recognized due to its simplicity, low cost and high enrichment, thus making it available to most analytical laboratories. Unfortunately, the lack of purification for samples with more complex matrices, such as fruits and vegetables, has caused this method to be limited to those with simpler matrices, specifically water and a few fruit juices. The QuEChERS extraction procedure is the most common technique for multi-residue pesticide analysis in foods, particularly fruits and vegetables [18]. The major drawback of this technique is its poor enrichment factor; the technique has higher detection limits compared with other techniques. Researchers have proposed a new method that consists of DLLME preconcentration after QuEChERS extraction [19–21]. Coupling these techniques takes advantage of the benefits of both methods while reducing some of the drawbacks. To the best of our knowledge, there are only a few reports on the extraction and enrichment of pesticide residues in agricultural products using the QuEChERSDLLME method [22–26]. Several sample preparation techniques, such as SPE [4,27], DLLME [28,29] and SPME [30], have been used for the extraction and cleanup of benzoylurea insecticides in fruits, vegetables, fruit juices and water. However, there are no published studies about the pretreatment of diflubenzuron and chlorbenzuron in fruits using the QuEChERS-DLLME method. The present study reports the development of a reliable and sensitive method for the simultaneous determination of two benzoylurea insecticides (diflubenzuron and chlorbenzuron) in grapes and pears. The method is based on liquid–liquid partitioning with acetonitrile and salts (QuEChERS) combined with an ionic-liquid-based dispersive liquid– liquid microextraction (IL-DLLME) followed by HPLC with diode array detection (DAD).
2 Materials and methods 2.1 Chemicals and reagents Diflubenzuron and chlorbenzuron standard solutions (100 mg/L, purity > 99%) were obtained from the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Agro-Environmental Protection Institution, Ministry of Agriculture (Tianjin, China). 1-Hexyl-3-methylimidazolium hexafluorophosphate ([HMIM][PF6 ], ࣙ99.0% purity), 1-butyl3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ], ࣙ99.0% purity), 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM][PF6 ], ࣙ99.0% purity) and 1-octyl-3methylimidazolium tetrafluoroborate ([OMIM][BF4 ], ࣙ99.0% purity) were purchased from Shanghai Cheng Jie Chemical, China. HPLC-grade solvents methanol and acetonitrile, primary secondary amine (PSA) and graphitized carbon black (GCB) were purchased from Shanghai ANPEL Scientific Instrument, China. An ultrapure water purification system with UV+UF optional accessories (UNIQUE-R20, Research, China) was used throughout this study. A 0.22 m cellulose membrane filter was used for filtration of the fruit samples. Stock solutions of diflubenzuron and chlorbenzuron were prepared at 100 mg/L in methanol and were stored in amber glass vials at –20⬚C. Serial dilutions of the standard working solutions were prepared in methanol.
2.2 Instruments and equipment An Agilent 1260 HPLC system (Agilent Technologies, Germany) was used for chromatographic analysis and consisted of a binary pump, automatic sample injector, degasser, and a diode array detector. The software was OpenLAB CDS ChemStation Edition Drivers Revision C.01.05. All separations were performed on a KR100-10 C18 column (5 m, 150 mm × 4.6 mm, Kromasil). The mobile phase consisted of the following gradient program: the initial mobile phase was 60:40 water/MeOH for 2 min, then a 22 min linear gradient from 60:40 to 15:85 water/MeOH, then a linear gradient from 15 to 10% water in 4 min, and the initial eluent composition was restored over a 4 min gradient and was maintained for 4 min. The injection volume was 20.0 L, and the flow rate was 1.0 mL/min. The detection wavelength was 258 nm. An ultrasonic cleaner (SCQ-2201B, Shanghai, China) and a model 5804R centrifuge (Eppendorf, Hamburg, Germany) were used in the experiment.
2.3 Samples Pear and grape samples were purchased from local supermarkets in Guangzhou, China. Samples were crushed homogeneously and stored at –20⬚C until analysis. For recovery determination, 10.0 g of the samples was spiked with different volumes of standard solutions and stored at room temperature for 2 h before analysis.
2.4 Extraction procedure Ten grams of homogenized fruit sample was weighed in a 50 mL PTFE centrifuge tube and extracted with 20 mL MeCN for 10 min in an ultrasonic cleaner. After the addition of 3 g of anhydrous sodium sulfate and 1.5 g of sodium chloride, www.jss-journal.com
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the mixture was vortexed for 1 min and then centrifuged at 3000 rpm for 5 min. A 10 mL sample of the upper MeCN layer was concentrated using a rotary evaporator at 40⬚C to dryness and was reconstituted with 1.6 mL of MeCN and transferred for clean-up by mixing with 100 mg of PSA and 150 mg of MgSO4 . The supernatant was collected after vortexing for 1 min and was centrifuged for 5 min at 3000 rpm. The sample was then filtered through a 0.22 m cellulose membrane filter before the DLLME step.
2.5 DLLME procedure A 1.1 mL aliquot of the acetonitrile extract solution that acted as the dispersive solvent for the DLLME was transferred into a centrifuge tube containing 90 L of ionic liquid [HMIM][PF6] (as the extraction solvent) and was then vortexed for 1 min. The mixture was rapidly injected into a 10 mL centrifuge tube containing 5 mL of 10% NaCl solution. This ternary component mixture was vortexed for 90 s to form an emulsified solution followed by centrifugation for 5 min at 3000 rpm. Finally, the upper aqueous phase was removed using a Pasteur pipette. The volume of the sedimented IL-phase (90 ± 3 L) was re-dissolved with 50 L of MeOH and was vortexed for 60 s. The solution was transferred to a sample vial, and 20 L was injected for HPLC analysis.
3 Results and discussion 3.1 Sample extraction and cleanup by QuEChERS A modified version of the QuEChERS method for sample preparation of fruits was used [31]. The procedure involves initial single-phase extraction of a 10 g sample with 20 mL of acetonitrile, followed by liquid–liquid partitioning by the addition of 3 g of anhydrous MgSO4 plus 1.5 g of NaCl. Normally, MeCN is a good extraction solvent for matrices with low fat content and does not significantly dissolve the matrix interfering compounds [32]. However, to facilitate the DLLME procedure, dispersive solid-phase extraction (DSPE), in which PSA was selected as the absorbent, was adopted to further purify the MeCN extract. In this procedure, different amounts of PSA (50, 100 and 200 mg) were tested. The recoveries obtained from the different PSA amounts were satisfactory (higher than 90%). However, the purification effect of 100 mg PSA was better than that of 50 mg PSA (Supporting Information Fig. S1). Ultimately, 100 mg of PSA was selected as the optimum quantity.
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[16, 33, 34]. When [BMIM][PF6 ] and [OMIM][BF4 ] were used as the extractants, there was limited or no sedimented phase formation, and progression to the next step was very difficult. However, when [HMIM][PF6 ] was used as the extraction solution, satisfactory extraction recoveries and enrichment factors were obtained (Fig. 1A and B). In the next step, the effect of the volume of [HMIM][PF6 ] on the extraction recoveries was investigated in the range of 80–120 L in 10 L intervals. As shown in Fig. 2A, the recoveries for diflubenzuron and chlorbenzuron varied from 72.9–90.2%. The highest recoveries for the two insecticides were obtained when 90 L of [HMIM][PF6 ] was used. Consequently, 90 L of [HMIM][PF6 ] was used as the optimum quantity. In our study, the solvent MeCN was used as the extraction solvent and then as the disperser solvent in the IL-DLLME step. To study the effect of the volume of dispersive solvent on the extraction efficiency, different volumes of QuEChERS extract, from 800–1200 L in 100 L increments, mixed with 90 L of extractive solvent were added to 5 mL of ultrapure water. As indicated by the results shown in Fig. 2B, higher extraction recoveries for the two insecticides were obtained when 1.1 mL of MeCN was used. Thus, 1.1 mL of the QuEChERS extract was chosen as the optimum volume for the dispersive solvent. IL-DLLME experiments were conducted using different amounts of NaCl (0, 5, 10 and 15%, m/v) under the same experimental conditions to test the salting-out effect. From the results shown in Fig. 3, increases of extraction recoveries for the two insecticides from 0–10% NaCl were observed, and the highest extraction recoveries were obtained at 10% w/v of NaCl. Therefore, 10% NaCl was added during the IL-DLLME procedure.
3.3 Matrix effects Different matrix components can be co-extracted with compounds of interest, causing matrix effects. Matrix components have an observable effect on the detection of target analytes by either enhancing or weakening their signal intensities. The matrix effects were evaluated by comparing the relative detector responses with the ratio of the calibration curves slopes obtained from the matrix-matched standard solutions and direct injection of MeOH solutions [35]. Matrix effects can be classified depending on the decrease/increase in the percentage of the slope: between ±20 and 0% represents a mild effect, between ±20 and ±50% is considered a medium effect and higher than ±50% is considered a strong effect of signal suppression or enhancement. The results did not show any matrix effects for the two pesticides (the ratios were close to 1.0 under all conditions).
3.2 Optimization of IL-DLLME To replace the highly toxic halogenated hydrocarbons used in previously reported DLLME methods, four ILs, namely, [HMIM][PF6 ], [BMIM][PF6 ], [OMIM][PF6 ] and [OMIM][BF4 ], were tested according to the methods in previous studies C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 Method validation Under the optimal conditions, the LODs, LOQs and linearity were investigated to validate the proposed method. Ten grams www.jss-journal.com
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Figure 1. Effect of different types of extractant on the recoveries (A) and enrichment factor (B) of BUs. Extraction conditions: 5.0 mL of water, 1.0 mL of MeCN, 0.1 mL of ILs and 0% NaCl.
Figure 2. Effect of the volume of [HMIM][PF6 ] (A) and MeCN (B) on the recoveries of BUs. Extraction conditions: (A) 5.0 mL of water, 1 mL of MeCN and 0% NaCl; (B) 5.0 mL of water, 90 L of [HMIM][PF6 ] and 0% NaCl. Table 1. Linear ranges, LODs, LOQs, average recoveries and repeatability (%RSD) obtained with the QuEChERS-IL-DLLME method in spiked fruits, analyzed by HPLC-DAD (n = 5 at each level) Pesticides
Fruits
Linearity equation
Linearity (g/mL)
r
MRLa ) (mg/kg) China
Diflubenzuron Chlorbenzuron
Grape Pear Grape Pear
y = 1959.4x – 1.1066 y = 1596.4x – 5.0874 y = 2260.9x – 4.1439 y = 1794.7x – 4.1542
0.01-0.5 0.01-0.5 0.01-0.5 0.01-0.5
0.9998 0.9996 0.9997 0.9995
c)
— 1 — —
LOD (mg/kg)
LOQ (mg/kg)
Japan 0.05 1 — —
0.005 0.005 0.005 0.005
0.01 0.01 0.01 0.01
Recovery (%) ± RSD (%) Level 1b)) 0.01
Level 2 0.05
Level 3 0.2
102.9 ± 0.5 101.3 ± 0.6 99.5 ± 1.0 102.9 ± 1.4
98.6 ± 1.0 107.2 ± 3.3 99.7 ± 1.93.4 100.9 ± 5.2
109.3 ± 0.6 106.8 ± 0.6 106.6 ± 3.4 101.3 ± 1.0
a) The data of MRLs from China Pesticide information Network. Available at . b) The unit of the concentration was mg/kg. c) No MRL be established at present.
of fruit samples spiked at five increasing concentration levels were subjected to the previously optimized methods described in Section 2.4 to obtain the calibration curves. The results are summarized in Table 1. The calibration graphs showed linearity for the concentration ranges of diflubenzuron and chlorbenzuron. Good correlation coefficients equal to or greater than 0.9995 were achieved for diflubenzuron and chlorbenzuron. The LODs were determined by injecting serial sample extraction solutions based on the S/N ratio of 3:1. The lowest fortified level was used as the LOQ. The LOD and LOQ for the two insecticides were 0.005 and 0.01 mg/kg, respectively. The LODs and LOQs of the present method were C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
below the MRLs established in China and Japan. The MRL of diflubenzuron was 0.05 mg/kg for grape and 1 mg/kg for pear in Japan. In China, the MRL of diflubenzuron for pear was 1 mg/kg. For chlorbenzuron, there are no MRLs for grape and pear in Japan and China (Table 1). Recovery assays were performed with the blank fruits samples (grape and pear) spiked at three concentration levels of 0.01, 0.05 and 0.2 mg/kg for diflubenzuron and chlorbenzuron. Each level was performed in five replicates as described in Section 2. The results are listed in Table 1. The relative recoveries for diflubenzuron and chlorbenzuron ranged from 98.6–109.3% at the three spiking levels, and the RSDs varied www.jss-journal.com
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Figure 3. Effect of the NaCl percentage on the recoveries of BUs. Extraction conditions: 5.0 mL of water, 90 L of [HMIM][PF6 ] and 1.0 mL of MeCN.
Figure 4. Typical chromatograms of BUs in spiked and blank fruit samples prepared by QuEChERS-IL-DLLME. (1) Diflubenzuron; (2) chlorbenzuron.
from 0.5–5.2%. Representative chromatograms of the BUs in the spiked and blank fruit samples from the supermarkets in Guangdong Province, China, are illustrated in Fig. 4.
traces of diflubenzuron or chlorbenzuron residues (data not shown). However, a more thorough and long-term investigation of fruit-derived BUs residues is necessary to ensure consumer safety.
3.5 Analysis of fruit samples The validated analytical method was used to monitor diflubenzuron and chlorbenzuron residues in ten fruit samples (five grapes and five pears) purchased from the local supermarkets in Guangdong Province, China. No samples showed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4 Comparison of QuEChERS-IL-DLLME with other methods Comparison of the presented QuEChERS-IL-DLLME method with several published methods demonstrated that the www.jss-journal.com
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Table 2. Comparison of the proposed methods and some other methods for the determination of benzoylureas residues in fruits samples
Method
Sample mass (g)
Organic solvent
LOD (g/kg)
Recovery (%)
Ref.
GPC-HPLC-UVD ME-SWATLD-HPLC-DAD SPE-HPLC-DAD PLE-LC-MS/MS QuEChERS-IL-DLLME-HPLC
50 50 25 5 10
Acetone (100 mL) +CH2Cl2 (50 mL) Methanol (50 mL) Acetonitrile (50mL) +(Petroleum ether+ Acetone) (65 mL) Ethyl acetate (25 mL) +Methanol (5 mL) Acetonitrile (20 mL)+ [HMIM][PF6] (90 L)
10–30 17–440 20–50 0.7–2 5
71–90 91–116 80–120 58–97 98–109
[36] [37] [38] [39] Proposed method
proposed method exhibited satisfactory extraction performance in terms of the relative recoveries and precisions (Table 2) [36–39]. At the same time, the LODs with QuEChERS-IL-DLLME were better than most of the other methods (data shown in Table 2). This can be explained by the fact that high enrichment factors could be obtained by DLLME. A high enrichment factor can also be obtained by increasing the quantity of analyzed samples and extraction solvent in conventional methods. However, this will also increase the consumption of organic solvents and the cost of the overall experiment. Additionally, the proposed method has the advantage of lower consumption of organic solvents (approximately 20 mL of organic solvent, data shown in Table 2). According to the comparison, the QuEChERS-ILDLLME method was concluded to be rapid, effective and environmentally friendly.
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5 Conclusions
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In this study, a novel analytical method, combining QuEChERS extraction with IL-DLLME procedures followed by HPLC–DAD, was successfully developed for the extraction, cleanup and preconcentration of diflubenzuron and chlorbenzuron in fruit samples. The combination of QuEChERS with DLLME provides an inexpensive sample pretreatment that ensures a high enrichment factor and low detection limits. The satisfactory extraction efficiency allows the determination of BUs below the maximum content allowed by China, even using DAD detection. QuEChERS combined with DLLME provides a useful method for the analysis of BUs in fruits for routine laboratory food quality analysis and safety control.
[13] Zgloa-Grzeskowiak, A., Grzeskowiak, T., TrAC, Trends Anal. Chem. 2011, 30, 1382–1399.
The authors have declared no conflict of interest.
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