Food Chemistry 133 (2012) 544–550

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Analytical Methods

Dispersive liquid–liquid microextraction combined with sweeping micellar electrokinetic chromatography for the determination of some neonicotinoid insecticides in cucumber samples Shuaihua Zhang, Xiumin Yang, Xiaofang Yin, Chun Wang, Zhi Wang ⇑ Key Laboratory of Bioinorganic Chemistry, College of Science, Agricultural University of Hebei, Baoding 071001, Hebei, China

a r t i c l e

i n f o

Article history: Received 20 June 2011 Received in revised form 10 January 2012 Accepted 14 January 2012 Available online 25 January 2012 Keywords: Dispersive liquid–liquid microextraction Sweeping Micellar electrokinetic chromatography Neonicotinoid insecticides Cucumber samples

a b s t r a c t A rapid, simple and sensitive method has been developed for the analysis of some neonicotinoid insecticides in cucumber samples by using dispersive liquid–liquid microextraction (DLLME) coupled with sweeping in micellar electrokinetic chromatography (MEKC). Under optimised conditions, the enrichment factors were achieved in the range from 4000 to 10,000. The linearity of the method was in the range from 2.7 to 200 ng g1 for thiacloprid, acetamiprid and imidacloprid, and in the range from 4.0 to 200 ng g1 for imidaclothiz in cucumber samples, with the determination coefficients (r2) ranging from 0.9924 to 0.9968. The limits of detection (LODs, S/N = 3) ranged from 0.8 to 1.2 ng g1. The relative standard deviations (RSDs) at the concentration levels of 10.0 and 50.0 ng g1 each of the neonicotinoid insecticides in cucumber samples varied from 3.8% to 6.3%. The developed method has been successfully applied to the analysis of the neonicotinoid insecticides in cucumbers with a satisfactory result. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Neonicotinoid insecticides are a relatively new group of active ingredients with novel modes of actions. These insecticides are active against numerous sucking and biting pests and insects, including whiteflies, aphides, beetles and some lepidoptera species as well (Meienfisch, Brandl, Kobel, Rindlisbacher, & Senn, 1999; Tomizawa & Casida, 2005). Neonicotinoid insecticides act as agonists at the insect nicotinic acetylcholine receptors (nAChRs), which plays an important role in synaptic transmission in the central nervous system (Muccio et al., 2006). They could give rise to serious risks for the health and safety of the consumers of the agricultural products due to their distribution on large areas of agricultural land. The amended European Union legislation has set the maximum residue limits (MRLs) for neonicotinoid insecticides in different agricultural products. The MRLs for fruit, vegetable and cereals were between 0.1 and 1.0 mg kg1 (Commission Directive 2007/ 11/EC). Therefore, the evaluation and monitoring of trace levels of these insecticides in vegetables is necessary and demands highly efficient, selective and sensitive analytical techniques. Different analytical techniques, including liquid chromatography–mass spectrometry (LC–MS) (Muccio et al., 2006; Obana, Okihashi, Akutsu, Kitagawa, & Hori, 2003; Seccia, Fidente, Montesano, & Morrica, 2005), liquid chromatography–tandem mass spec⇑ Corresponding author. Tel./fax: +86 312 7521513. E-mail addresses: [email protected], [email protected] (Z. Wang). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.028

trometry (LC–MS/MS) (Radišic´, Grujic´, Vasiljevic´, & Lauševic´, 2009; Xiao, Li, Wang, Shen, & Ding, 2011), high-performance liquid chromatography with diode-array detection (HPLC-DAD) (Obana, Okihashi, Akutsu, Kitagawa, & Hori, 2002; Watanabe, Baba, & Eun, 2007; Wu et al., 2011), gas chromatography-mass spectrometry (GC–MS) (Rossi, Sabatini, Cenciarini, Ghini, & Girotti, 2005), and enzymelinked immunosorbent assays (ELISAs) (Watanabe, Miyake, Baba, Eun, & Endo, 2006) have been reported for the determination of neonicotinoid insecticides in various types of samples. More recently, capillary electrophoresis (CE), because of its high separation efficiencies, short analysis time, small sample consumptions and low operation cost, has also been used for pesticide residue analysis (Zhu & Lee, 2001). However, when the most popular CE photometric detector is used, the main disadvantage of CE is its poor concentration sensitivity due to the short optical length of the capillary (Simpson, Quirino, & Terabe, 2008). This shortcoming has prevented CE from being more widely used for pesticide residues analysis. To overcome this sensitivity problem, several on-line preconcentration strategies, with the advantages of simplicity and economy, have been developed to increase the sensitivity of CE, such as field amplification (Chien & Burgi, 1992), dynamic pH junction (Britz-McKibbin, Bebault, & Chen, 2000), transient isotachophoresis (tITP) (Beckers & Bocˇek, 2000) and sweeping (Quirino & Terabe, 1998). Sweeping is an effective on-line sample concentration technique in micellar electrokinetic chromatography (MEKC). It consists of the introduction of a large sample zone prepared in a matrix devoid of pseudostationary phase, wherein the analytes are picked-up and accumulated by the pseudostationary phase that

S. Zhang et al. / Food Chemistry 133 (2012) 544–550

penetrates the sample. This technique has been successfully applied for the on-line preconcentration of aromatic amines (Quirino, Iwai, Otsuka, & Terabe, 2000), phenoxy acid herbicides (Quirino, Terabe, Otsuka, Vincent, & Vigh, 1999), quaternary ammonium herbicides (Núñez, Kim, Moyano, Galceran, & Terabe, 2002), triazine herbicides (da Silva, de Lima, & Tavares, 2003), phenolic compounds (Huang, Lien, & Huang, 2006), carbamate pesticides (Zhang et al., 2010) and other pesticides (Breadmore, Dawod, & Quirino, 2011; El Deeb, Iriban, & Gust, 2011; See, Hauser, Ibrahim & Sanagi, 2010). Prior to the instrumental determination of the residues, extraction and preconcentration of the sample is often required. For the preconcentration and cleanup of the neonicotinoid insecticides, liquid–liquid extraction (LLE) (Watanabe et al., 2006) and solidphase extraction (SPE) (Obana et al., 2002, 2003; Seccia et al., 2005; Muccio et al., 2006; Watanabe et al., 2007) are the most commonly used techniques. However, LLE suffers from the disadvantage of requiring large amount of both samples and toxic organic solvents. SPE techniques typically require reduced amounts of organic solvents relative to LLE, but SPE sometimes suffers from analytes breakthrough when large sample volumes are analysed. Moreover, both techniques are tedious, time-consuming and expensive. To overcome these shortcomings in LLE and SPE, in recent years, extensive efforts have been made to the development of new sample preparation techniques that can save time, labour and solvent consumption and, therefore, can improve the analytical performance of the procedure. Dispersive liquid–liquid microextraction (DLLME), which was first reported by Rezaee and co-workers in 2006 (Rezaee et al., 2006), can overcome some of the abovementioned limitations with the advantages of simplicity of operation, rapidity, low cost and high enrichment factor (Fattahi, Assadi, Hosseini, & Jahromi, 2007; Nagaraju & Huang, 2007; Wu, Wang, Liu, Wu, & Wang, 2009). DLLME is based on the formation of the fine droplets of an extractant in an aqueous sample solution when a water-immiscible extraction solvent (extractant) dissolved in a water-miscible organic dispersive solvent is rapidly injected into the aqueous sample solution. The analytes in the sample solution are extracted into the fine droplets, which are further separated by centrifugation, and the enriched analytes in the sedimented phase are determined by either chromatographic or spectrometric methods. However, until now, there are very few literature reports about the applications of DLLME in combination with capillary electrophoresis for the analysis of organic pollutants in real samples. Therefore, the exploration of the potential applications of the DLLME technique in combination with CE for the analysis of more complex matrix samples, such as fruits and vegetables, is very desirable. Previously, we have reported a new strategy to apply DLLME procedure with sweeping MEKC (DLLME-sweeping-MEKC) for the analysis of some carbamate pesticides in apples (Zhang et al., 2010). In continuation to our previous endeavours, herein, we explore the DLLME-sweeping-MEKC method for the determination of some neonicotinoid insecticides in cucumber samples. Thiacloprid, acetamiprid, imidaclothiz and imidacloprid, which are most widely used in the local area, were selected as the analytes. As a result, the sensitivity of the analysis was much improved and satisfactory analytical results were achieved.

2. Experimental 2.1. Reagents, chemicals and materials Thiacloprid, acetamiprid, imidaclothiz and imidacloprid (all >99%) were purchased from Agricultural Environmental Protection Institution (Tianjin, China). Sodium dodecyl sulphate (SDS) was

545

purchased from Sigma–Aldrich (St. Louis, MO, USA). Boric acid (H3BO3), hydrochloric acid (HCl), sodium hydroxide (NaOH, 98%), acetonitrile, acetone, ethanol and methanol (HPLC-grade) were from Sinopharm Chemical Reagent Co., (Beijing, China). Dichloromethane (CH2Cl2), chloroform (CHCl3), carbon tetrachloride (CCl4), 1,2-dichloroethane (C2H4Cl2), 1,2-dichlorbenzene (C6H4Cl2) and chlorobenzene (C6H5Cl) were purchased from Beijing Chemical Reagent Co. (Beijing, China). All the solvents were filtered through a 0.45 lm MicroScience membrane filter from Tianjin Automatic Science Instrument Co., (Tianjin, China). The pH of H3BO3 solutions was adjusted with 1.0 mol l1 HCl. The background solution (BGS) was newly prepared everyday and sonicated for 5 min prior to use. Cucumber samples were purchased from local supermarket (Baoding, China). The mixture stock standard solution containing 10.0 lg ml1 each of the neonicotinoids was prepared in methanol and stored in glass-stoppered bottles at 4 °C. A series of standard solutions were prepared by mixing an appropriate amount of the stock solution with 150 mmol l1 H3BO3 (pH 4.7) after dryness under a stream of nitrogen. 2.2. Apparatus All CE experiments were performed on a Beckman P/ACE MDQ Capillary Electrophoresis System (Beckman Coulter, Fullerton, CA, USA) equipped with an auto sampler and a diode array detector (DAD). An uncoated fused-silica capillary (Yongnian Ruifeng Optical Fibre Factory, Hebei, China) of 50 cm (effective length, 40 cm) 75 lm i.d was used throughout the experiments. All of the operations were computer-controlled using Beckman P/ACE MDQ 32 karat software. 2.3. Sample preparation After homogenisation with a laboratory homogenizer, a 20.0 g portion of the homogenised cucumber sample was accurately weighed, put into a 20-ml centrifuge tube and diluted to 20.0 ml with double-distilled water. Then, the sample was centrifuged at 3500 rpm for 10 min. A 5.0 ml aliquot of the above supernatant was then transferred to a 10.0 ml screw cap glass tube with conic bottom. Then, 0.8 ml of acetonitrile (as dispersive solvent) containing 100.0 ll of CHCl3 (as extraction solvent) was rapidly added into the tube. After vortexing for 1 min, a cloudy solution that consisted of very fine droplets of CHCl3 dispersed into the aqueous sample was formed, and the analytes were extracted into the fine droplets. After centrifugation at 3500 rpm for 5 min, the CHCl3 phase was sedimented at the bottom of the centrifuge tube. The sedimented phase (about 90 ll) was completely transferred to another 1.0 ml conical bottom vial using a 100.0 ll microsyringe, evaporated to dryness under a mild nitrogen stream, and finally reconstituted with 20.0 ll 150 mmol l1 H3BO3 (pH 4.7) for CE analysis. 2.4. General electrophoresis procedure New capillaries was conditioned prior to use with 0.1 mol l1 NaOH (10 min), water (10 min), methanol (10 min), and water (5 min). To ensure repeatability, the capillary was flushed between consecutive analyses with 0.1 mol l1 NaOH at 20 psi for 3 min, then with double-distilled water for 3 min, and finally with the BGS for 5 min. For sweeping, sample was prepared in 150 mmol l1 H3BO3 (pH 4.7). The BGS was 50 mmol l1 H3BO3 (pH 2.0) containing 80 mmol l1 SDS and 25% methanol. The sample was introduced into the capillary by hydrodynamic injection at 0.5 psi for 90 s. Electrophoresis was performed at a constant voltage of 20.0 kV

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at 25 °C, with DAD detection at 243 nm for thiacloprid and acetamiprid, and 268 nm for imidaclothiz and imidacloprid, respectively. 3. Results and discussion 3.1. Optimisation of the sweeping conditions Sweeping is an on-line sample concentration technique in MEKC that relies on the sample matrix prepared in a buffer solution with conductivity that is similar, lower or higher than BGS but without adding pseudostationary phase (micelles). When charged micelles in BGS penetrate the sample zone during the application of voltage, picking or accumulating of the analytes occurs due to partitioning or interaction of the analytes with the micelles. Therefore, the analytes zones are narrowed due to a partitioning mechanism as the sample molecules are incorporated into the micelles phase. In this experiment, an aqueous solution containing 5.0 lg ml1 each of the neonicotinoid insecticides was used to study the sweeping performance under different experimental conditions, such as buffer concentration, buffer pH, SDS concentration, organic solvent content and injection time. 3.1.1. Effect of the buffer and SDS concentration Buffer concentration has a significant effect on the CE separation because it can influence the Joule heating, the electro-osmotic flow (EOF), and the current produced in the capillary. To obtain the best separation of the four neonicotinoids, the influence of the H3BO3 buffer concentration on the separation was investigated by changing the H3BO3 concentration to 5, 10, 25, 50, 80 and 100 mmol l1, respectively, while other experimental conditions remained unchanged (80 mmol l1 SDS and 25% methanol in the buffer). The result demonstrated that the migration time was slightly decreased with increased concentration of H3BO3. On the other hand, when the concentration of H3BO3 was lower than 25 mmol l1, the resolution of the analytes, especially for acetamiprid and imidaclothiz, was poor, and when the concentration of H3BO3 was more than 80 mmol l1, the baseline noise was increased because the effect of Joule heat became more pronounced. As a result, 50 mmol l1 H3BO3 was selected for subsequent investigations. Different concentrations of the surfactant SDS (20, 40, 60, 80, 100, and 120 mmol l1) were tested to observe the effect of the concentration of SDS in the BGS. The result indicated that both the heights and areas of the neonicotinoid peaks were increased but the resolution of the analytes decreased with increased SDS concentration. When the concentrations of SDS were higher than 80 mmol l1, the peaks of the neonicotinoids became broadened. Giving an overall consideration of both sensitivity and resolution, 80 mmol l1 SDS was selected for further studies.

neonicotinoids were broad, and when the pH was higher than 2.2, the resolution of the four neonicotinoids became deteriorated, particularly for acetamiprid and imidaclothiz. Consequently, pH 2.0 was chosen for further optimisations. 3.1.3. Effect of organic modifier concentration The addition of organic solvents, such as acetonitrile or methanol, to the BGS could influence both the resolution and migration time of the analytes since they could cause a difference in affinity between micelles and analytes. In this experiment, the effect of methanol concentration was investigated by changing its concentration at 5%, 10%, 15%, 20%, 25% and 30% (v/v), respectively. The results are shown in Fig. 1. When the methanol concentration was increased, the resolutions of the analytes were improved but with increased migration time. The separation between acetamiprid and imidaclothiz was poor when methanol concentration was lower than 20%. But when the methanol concentration was higher than 30%, the migration time for both imidaclothiz and imidacloprid was longer than 20 min. Giving an overall consideration of both the resolution and migration time, 25% of methanol was selected for the experiment. 3.1.4. Effect of sample injection length For sweeping technique, the injected length of an analyte zone is theoretically narrowed by a factor equal to 1/(1 + k), where k is the retention factor. In other words, high k analytes could be concentrated without bounds. If the k value of the analyte is not sufficiently high, it is proposed that the injected sample plug length must be optimised (Zhu, Tu, & Lee, 2002). In this study, with the BGS of 50 mmol l1 H3BO3 containing 80 mmol l1 SDS and 25% methanol, the sample solution was injected into the capillary at 0.5 psi for 5, 10, 60, 90, 120 and 180 s, corresponding to the injection length of 0.6, 1.2, 7.3, 10.9, 14.6, and 21.9 cm, respectively. As a result, with the increase of the injected sample plug length, the 300

1

2

D 250

1

C

200

2 3

4

1 2

150

B 3 4 1

100

2 3.1.2. Effect of the BGS pH The acidity of the running buffer affects the migration time and separation efficiency of the analytes. With negative polarity at low pH, the basic neonicotinoids were cationic and the migration direction of both the EOF and the cationic neonicotinoids was opposite to that of the SDS micelles. Since the electrophoretic velocity of SDS (lep(SDS)) was greater than the EOF, the analytes were brought to the detector with the aid of the SDS micelles. In this study, the running buffers of 50 mmol l1 H3BO3 containing 80 mmol l1 SDS and 25% methanol at different pH values (1.5, 1.8, 2.0, 2.2, 2.5, and 3.0) were examined for the optimisation. It was found that with the increase of BGS pH, the migration time of the analytes was decreased and the peaks of the analytes became sharper; on the other hand, when pH was lower than 1.8, all the peaks of the

A

50

34

0 0

5

10

15

20

Minutes

Fig. 1. Effect of the concentration of methanol in BGS on the on-line sweeping MEKC. The BGS consisted of 50 mmol l1 H3BO3 (pH 2.0) containing 80 mmol l1 SDS and methanol content at (A) 15%, (B) 20%, (C) 25% and (D) 30%. The sample (5.0 lg ml1 of each neonicotinoid: 1-thiacloprid, 2-acetamiprid, 3-imidaclothiz and 4-imidacloprid) was prepared in 150 mmol l1 H3BO3 (pH 4.7); DAD monitoring wavelength: 243 nm.

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3.1.5. Choice of sample matrix In the early work of sweeping, the conductivity of the sample matrix was usually adjusted to be nearly equal to that of the BGS. Therefore, homogeneous electric field strength was assumed throughout the whole capillary under sweeping conditions, which is different from field-enhanced stacking techniques (Quirino & Terabe, 1999; Simpson et al., 2008). Later, Palmer et al. reported that the sample solution devoid of micelles with a conductivity of two to three times greater than that of BGS could improve the focusing effect in MEKC (Palmer, Munro, & Landers, 1999). In this study, to examine the influence of the sample matrix on the separation of the analytes, the sample matrix effect was examined by dissolving the analytes in different H3BO3 buffers with the same pH at 4.7 but with different conductivities or concentrations of H3BO3, i.e., 10, 20, 50, 100, 150, and 200 mM, respectively. As a result, the focusing effect in sweeping-MEKC was improved as the concentration of H3BO3 was increased from 10 to 150 mM and then remained almost unchanged. Therefore, 150 mM H3BO3 was chosen. When the concentration of H3BO3 was 150 mM, the conductivity of the sample matrix was about three times greater than that of BGS. This result is in agreement with those reported by Palmer et al. (1999). Based on the above optimisations, the optimal experimental conditions for sweeping MEKC were selected as follows: BGS was 50 mmol l1 H3BO3 (pH 2.0) containing 80 mmol l1 SDS and 25% methanol; sample was prepared in 150 mmol l1 H3BO3 (pH 4.7); sample injection was performed at 0.5 psi for 90 s. Under these optimised conditions, compared with normal hydrodynamic sample injection in MEKC (0.5 psi, 5 s), the sensitivity enhancement factors (SEFs) (Quirino & Terabe, 1998; Zhang et al., 2010; Zhu et al., 2002) of the sweeping MEKC in terms of peak height (SEFH) for thiacloprid, acetamiprid, imidaclothiz and imidacloprid were 63, 42, 32, and 36, and in terms of peak area (SEFA), were 61, 40, 35, and 43, respectively. 3.2. Optimisation of DLLME In order to optimise the DLLME procedure, 5.0 ml double-distilled water spiked with 50 ng ml1 each of the four neonicotinoid insecticides was used to study the extraction performance of the DLLME under different experimental conditions. All the experiments were performed in triplicate, and the means of the results were used for optimisation. For DLLME, extraction recovery (R%) was calculated according to the following equation:

R% ¼

V rec C rec  100 C 0 V aq

where R%, Crec, Vrec, C0 and Vaq are the extraction recovery, the analytes concentration in the final reconstituted solution in the extraction, the volume of the final reconstituted solution, the initial analytes concentration in the aqueous samples and the volume of the aqueous sample, respectively (Fattahi et al., 2007; Nagaraju & Huang, 2007; Wu et al., 2009).

meet some requirements. It should have a higher density than water, a low solubility in water, and high extraction capability for the target analytes, and also should form a stable two-phase system in the presence of a dispersive solvent when injected to an aqueous solution. Among the solvents with density higher than water (mainly halogenated hydrocarbons), six extraction solvents including CCl4, CHCl3, C2H4Cl2, CH2Cl2, C6H4Cl2, and C6H5Cl were investigated for the extraction. On the other hand, the selection of a dispersive solvent is limited to the solvents such as acetonitrile, acetone and ethanol, which are miscible with both water and extraction solvents. In this study, all combinations of using CCl4, CHCl3, C2H4Cl2, CH2Cl2, C6H4Cl2, and C6H5Cl as extraction solvents with acetonitrile, acetone and ethanol as dispersive solvents were tested. As a result, in the case of CH2Cl2 and C2H4Cl2, a twophase system was not observed with any of the dispersive solvents tested. For CCl4, the extraction recoveries of the four neonicotinoids were relatively lower than other extraction solvents. Based on the above results, CHCl3, C6H4Cl2 and C6H5Cl were chosen as potential extraction solvents for further study. Fig. 2 shows the effect of the extraction solvents (CHCl3, C6H4Cl2 and C6H5Cl) on the extraction recoveries with acetonitrile as dispersive solvent. As can be seen from Fig. 2, CHCl3 gives the highest extraction efficiency for all the analytes investigated. Therefore, CHCl3 was selected as the extraction solvent for subsequent experiments. On the other hand, the effects of different dispersive solvents on the extraction ability of the analytes were also studied. As a result, the highest extraction ability was achieved when acetonitrile was used as a dispersive solvent. Consequently, acetonitrile was selected as the dispersive solvent. 3.2.2. Effect of extraction solvent volume To study the effect of the extraction solvent volume on the performance of the presented DLLME procedure, different volumes of CHCl3 (40.0, 60.0, 80.0, 100.0, and 150.0 ll) with a constant volume of the dispersive solvent acetonitrile (0.8 ml) were investigated. The results exhibited that the extraction recovery was increased with increased volume of CHCl3 from 40.0 to 100.0 ll; after that, the extraction recoveries of the neonicotinoids remained almost constant. Therefore, 100.0 ll of CHCl3 was selected. 3.2.3. Effect of dispersive solvent volume For the optimisation of the dispersive solvent volume, the experiments were performed by using different volumes (0.5, 0.8, 1.0, 1.2 and 1.5 ml) of the dispersive solvent. The results indicated that with increased volume of acetonitrile, the extraction efficiency was increased first, and then decreased for all the analytes. The reason for this could be that at a low volume of acetonitrile, a cloudy state was not formed well, thus giving a low recovery; at a 35

Thiacloprid Acetamiprid Imidaclothiz Imidacloprid

30 Extraction recovery/ %

peak heights were increased, but the resolutions between the peaks were gradually deteriorated. When the injection time was more than 120 s, the separations of all the peaks were greatly decreased. As a compromise between the resolution and sensitivity, the injection time of 90 s at 0.5 psi (10.9 cm, 21.8% of the capillary length) was chosen as the optimum condition.

25 20 15 10 5 0

C6H5Cl

3.2.1. Selection of the extraction and dispersive solvent Choosing an appropriate extraction solvent is of primary importance for most extractions. In DLLME, the extraction solvent has to

CHCl3

C6H4Cl2

Fig. 2. Effect of different extraction solvents on the extraction recovery of the neonicotinoids. Extraction conditions: sample volume, 5.0 ml; dispersive solvent, 0.8 ml acetonitrile; extraction solvent volume, 60 ll.

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Table 1 Sensitivity enhancement factors of the pesticides for DLLME alone, on-line sweeping-MEKC alone, and the combination of the DLLME with sweeping-MEKC. Insecticides

Thiacloprid Acetamiprid Imidaclothiz Imidacloprid

DLLME

Sweeping-MEKC

DLLME-Sweeping-MEKC

EF

%RSD

SEFH

%RSD

SEFA

%RSD

SEFH

%RSD

SEFA

%RSD

177 130 127 111

4.5 4.2 3.7 5.8

63 42 32 36

2.9 3.8 1.8 3.1

61 40 35 43

2.8 3.5 4.2 4.0

9918 4971 4016 3985

3.3 7.2 6.3 5.9

9957 5198 4320 4590

4.8 6.9 7.0 5.4

Table 2 Within laboratory reproducibilities (RSDs), linear range (LR), r2, F and limits of detection (LODs) of the method for neonicotinoid insecticides in cucumber samples by the DLLME with sweeping-MEKC. Insecticides

Thiacloprid Acetamiprid Imidaclothiz Imidacloprid a

LR (ng g1)

2.7–200 2.7–200 4.0–200 2.7–200

r2

0.9968 0.9966 0.9962 0.9924

F (6.61)

a

9.92 9.36 8.90 7.39

LODs (ng g1)

0.8 0.8 1.2 0.8

LOQs (ng g1)

2.7 2.7 4.0 2.7

RSD (%) Intraday (n = 5)

Interday (n = 15)

4.4 3.9 3.8 4.0

6.5 6.4 4.5 7.1

The number in the parenthesis is the theoretical F value (P = 0.05).

higher volume of acetonitrile, the solubility of the insecticides in water was increased, leading to a decreased extraction efficiency because of a decrease of the distribution coefficient. Based on the experimental results, 0.8 ml of acetonitrile was chosen. 3.2.4. Effect of extraction time In DLLME, the extraction time is defined as the interval between the addition of the mixture of the extraction solvent (CHCl3) and dispersive solvent (acetonitrile) to the sample and the start of centrifugation. The effect of extraction time was studied over the time range between 1 and 10 min (the mixture solution was first vortexed for 1 min and then was shaken in a shaker for an appropriate time). The results indicated that the extraction time has no influence on the extraction efficiencies. This could be attributed to the fact that equilibrium state can be achieved very quickly in DLLME and consequently, the extraction time required is very short. In this experiment, vortexing for 1 min was adopted. 3.2.5. Effect of ionic strength The influence of ionic strength on the performance of DLLME was investigated by adding different amounts of NaCl (0–10%, w/ v). The results exhibited that the salt addition had no significant effect on the extraction recoveries when the concentration of NaCl was changed from 0% to 6%. However, when the NaCl concentration was higher than 8%, the extraction solvent phase could not be sedimented at the bottom of the centrifuge tube, but went to the upper layer in the tube. Based on the above experimental results, no salt was added in the subsequent experiments. Under the above optimised experimental conditions, the enrichment factors (EF) of DLLME for thiacloprid, acetamiprid, imidaclothiz and imidacloprid were 177, 130, 127 and 111, respectively.

DLLME-sweeping MEKC method provided about 4000–10,000-fold sensitivity enhancement without obvious loss of resolution. The above results demonstrated that the developed DLLME-sweeping MEKC method indeed has markedly improved the detection sensitivity compared with conventional MEKC. 3.4. Method evaluation Neonicotinoids-free samples were used as blanks for matrixmatched standard calibrations. An appropriate amount of the mixture standard solution of the neonicotinoid insecticides was added into the homogenised cucumber samples. A series of working samples containing each of the neonicotinoids at seven concentration levels of 2.0, 5.0, 10.0, 20.0, 50.0, 100.0 and 200.0 ng g1 were prepared for the establishment of the calibration curve. For each level, five replicate extractions were performed and the peak areas of the analytes were used as quantification signals. The linear range, determination coefficients (r2), the limits of detection (LODs, S/ N = 3), the limits of quantification (LOQs, S/N = 10) are summarised in Table 2. The LODs were between 0.8 and 1.2 ng g1 and LOQs between 2.7 and 4.0 ng g1. The signal was linear over the concentration range from their corresponding LOQs to 200 ng g1 for all the four neonicotinoid insecticides in cucumbers, with r2 ranging from 0.9924 to 0.9968. F-test was applied to the seven different concentrations of the analytes for linear equations. The results (see Table 2) show that all of the calculated F values for the four Table 3 Determinations of the neonicotinoid residues in spiked cucumber samples and the recoveries of the method.

3.3. Sensitivity enhancements of the combination of the DLLME with sweeping-MEKC The sensitivity enhancement factors of the DLLME coupled with sweeping-MEKC were evaluated according to the equations reported in our previous work (Zhang et al., 2010) in the form of both SEFH (SEF in terms of peak height) and SEFA (SEF in terms of peak area). The comparisons between the DLLME alone, sweeping MEKC alone, and this combined DLLME-sweeping MEKC method in terms of SEFH and SEFA are summarised in Table 1. Compared with normal hydrodynamic sample injection in MEKC (0.5 psi, 5 s), the

Spiked (ng g1)

Thiacloprid

0 10 50

5.90 13.87 52.10

79.7 92.4

6.3 4.9

0 10 50

4.31 14.11 52.95

98.0 97.3

5.5 5.3

8.30 43.65

83.0 87.3

3.8 4.6

nd 8.73 48.10

87.3 96.2

4.3 5.1

Acetamiprid

Imidaclothiz

Imidacloprid

a b

0 10 50 0 10 50

R: recovery of the method. nd: not detected.

Measured (ng g1)

a

Insecticides

nd

R

(%)

RSD (%)

b

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15

15

u

A u

mAU

10

B

10

5

u

5

4 1

u 1 u

0

0

5

10

u 2

uu

2 3

0

15

0

5

10

15

time/ min

time/ min

Fig. 3. The electropherograms of the cucumber sample (A) and the cucumber sample spiked with each neonicotinoid at 15.0 ng g1 (B). Extraction and separation conditions are the same as described in the section of Experimental. Peak identifications are the same as in Fig. 1, and u represents unidentified peaks. DAD monitoring wavelength: 268 nm.

neonicotinoids were higher than the theoretical F value 6.61 (P = 0.05). Therefore, the linearity of the DLLME-sweeping-MEKC method was significant according to the Analysis of Variance (ANOVA) at the 95% confidence level. The within laboratory reproducibilities of the developed DLLMEsweeping-MEKC method was evaluated in terms of intraday and interday precisions by extracting and determining the analytes from the spiked cucumber samples at the concentration of each insecticide at 15.0 ng g1 in the same day and on three consecutive days. The results, expressed as the relative standard deviations (RSDs) of peak areas are also presented in Table 2. As can be observed, an acceptable precision was obtained with intraday RSD values below 4.4% and interday RSD values within 7.1%. These results indicate that the developed method is sensitive and repeatable.

developed by combining the DLLME with the on-line preconcentration procedures of sweeping-MEKC. The results demonstrate that the method has high enrichment factors, good recoveries and within laboratory reproducibilities with a short analysis time. The DLLME-sweeping MEKC method has been successfully applied to the analysis of thiacloprid, acetamiprid, imidaclothiz and imidacloprid in cucumbers, indicating that the method is suitable for the determination of the neonicotinoid insecticides in real samples.

3.5. Sample analysis

References

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Acknowledgments Financial support from the National Nature Scientific Foundation of China (No. 31171698) is gratefully acknowledged.

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