2380 Pei Liang Jinjin Wang Guojiao Liu Jinyan Guan Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, P. R. China Received April 20, 2014 Revised June 7, 2014 Accepted June 11, 2014
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Research Article
Determination of four sulfonylurea herbicides in tea by matrix solid-phase dispersion cleanup followed by dispersive liquid–liquid microextraction Matrix solid-phase dispersion combined with dispersive liquid–liquid microextraction has been developed as a new sample pretreatment method for the determination of four sulfonylurea herbicides (chlorsulfuron, bensulfuron-methyl, chlorimuron-ethyl, and pyrazosulfuron) in tea by high-performance liquid chromatography with diode array detection. The extraction and cleanup by matrix solid-phase dispersion was carried out by using CN-silica as dispersant and carbon nanotubes as cleanup sorbent eluted with acidified dichloromethane. The eluent of matrix solid-phase dispersion was evaporated and redissolved in 0.5 mL methanol, and used as the dispersive solvent of the following dispersive liquid–liquid microextraction procedure for further purification and enrichment of the target analytes before high-performance liquid chromatography analysis. Under the optimum conditions, the method yielded a linear calibration curve in the concentration range from 5.0 to 10 000 ng/g for target analytes with a correlation coefficients (r2 ) ranging from 0.9959 to 0.9998. The limits of detection for the analytes were in the range of 1.31–2.81 ng/g. Recoveries of the four sulfonylurea herbicides at two fortification levels were between 72.8 and 110.6% with relative standard deviations lower than 6.95%. The method was successfully applied to the analysis of four sulfonylurea herbicides in several tea samples. Keywords: Dispersive liquid–liquid microextraction / High-performance liquid chromatography / Matrix solid-phase dispersion / Sulfonylurea herbicides / Tea samples DOI 10.1002/jssc.201400449
1 Introduction Tea is one of the most consumed nonalcoholic beverages worldwide and valued for its specific aroma and flavor as well as potential health-promoting properties [1]. China is one of the largest tea-producing and exporting countries in the world. To control pest and disease, pesticides are widely used in nearly every period of cultivation, storage, and product manufacturing processes of tea. Pesticides residue may be present in tea and cause health threats to human through tea consumption [2]. In order to protect the health of consumers and regulate the international tea trade, several countries, and international organizations have defined maximum residue limits of pesticides for tea [3]. As the maximum residue limits have become stricter and comprehensive, the development Correspondence: Dr. Liang, Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Guizhishan, Wuhan, HuBei 430079, China E-mail: [email protected]
Abbreviations: DLLME, dispersive liquid–liquid microextraction; MSPD, matrix solid-phase dispersion; MWNT, multiwalled carbon nanotube; SUH, sulfonylurea herbicide
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of sensitive, accurate, and rapid methods for the analysis of pesticides in tea has become an important and urgent issue. The tea samples consist of complex matrix components including pigments, alkaloids and polyphenols, and lipophilic inclusions [4]. During the extraction process of pesticides residue analysis, they could be extracted with organic solvent that may lead to serious difficulties in qualitative and quantitative analysis of the analytes. In order to ensure the accuracy and reliability of experimental results, a sample pretreatment is often required not only to isolate and concentrate the target analytes, but also to reduce or eliminate the huge matrix effects caused by coextractives in tea extracts. The conventional methods of sample preparation for pesticides in tea include ultrasonic extraction, gel permeation chromatography, mechanical shaking extraction, and SPE. These techniques are easy to carry out and provide good recoveries. Nevertheless, they are time consuming, labor intensive, consume a high amount of organic solvent and have poor repeatability [5]. Dispersive liquid–liquid microextraction (DLLME) is a novel sample pretreatment method based on a ternary component solvent system [6]. In this method, water-immiscible extraction solvent dissolved in a water-miscible dispersive solvent is rapidly injected into an aqueous solution by syringe, and a cloudy solution is formed. The analyte in the sample is
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Figure 1. Chemical structure and pKa values of the studied sulfonylurea herbicides.
extracted into the fine droplets of extraction solvent. After extraction, phase separation is performed by centrifugation, and the enriched analyte in the sediment phase is determined by chromatography or spectrometry methods. The advantages of the DLLME method are simplicity of operation, rapidity, low organic solvent consumption, high recovery, and enrichment factor [7]. This method has been applied to determine a variety of organic pollutants and metal ions in environmental samples [8–11]. However, due to its lack of selectivity, DLLME is confronted with difficulty of extracting target analytes from complex matrix samples [12]. This is the main reason that the most reported applications of DLLME have been focused on simple matrix samples. Therefore, the exploration of the applications of DLLME technique for more complex matrix samples is desirable. The combination of DLLME with a sample cleanup method, such as SPE, dispersive SPE, and microwave-assisted extraction, has been reported [13–15]. One of the advantages of such a combination is that it can be used for complex matrix samples. Matrix solid-phase dispersion (MSPD) was introduced in 1989 by Barker for conducting simultaneous disruption and extraction of solid, semisolid, and highly viscous samples [16]. This method enables the simultaneous accomplishment of both extraction and cleanup steps, and has been demonstrated to be an attractive alternative to sample preparation of complex matrices. MSPD is achieved mainly through the dissolution and dispersion of the organic phase bound to the sorbent instead of solvent extraction, and thus it consumes far less organic solvent and requires a shorter extraction time compared with conventional extraction methods [17]. The application of MSPD has shown satisfactory results in the extraction of pesticides, antibiotics, additives, banned dyes, and other pollutants from a wide variety of complex samples [18–22]. To our knowledge, there are limited papers in the literature reporting the combination of MSPD with DLLME [23–25]. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The aim of this work was to develop a new sample pretreatment method by combining MSPD with DLLME, thus to enhance the selectivity of the DLLME method. In this study, the applicability of the proposed method was explored for the extraction of some sulfonylurea herbicides (SUHs) in tea samples prior to their analysis by HPLC. SUHs are used worldwide to control several broad-leaved weeds and some grasses in a variety of crops because of their low application rates, unprecedented herbicidal activity, and low mammalian toxicity [26, 27]. The mode of action of SUHs is inhibition of the biosynthesis of branched chain amino acids [28], and SUHs residues could stop cell division in plants and lead to yield reduction in sensitive crops [29, 30]. The maximum residual limit of SUHs in tea (0.05 mg/kg) was established by the European Union [31]. Therefore, efficient, reliable and sensitive analytical methods are indispensable for the control of SUHs residues. Four of the most widely used SUHs (chlorsulfuron, bensulfuron-methyl, chlorimuron-ethyl, and pyrazosulfuron, the structures of these molecules are shown in (Fig. 1)) in the local area were chosen as target analytical compounds. The factors affecting the detection sensitivity of the method are investigated in detail.
2 Materials and methods 2.1 Standards, reagents, and samples Chlorsulfuron (99.5%), Bensulfuron-methyl (99.0%), Chlorimuron-ethyl (99.0%), and Pyrazosulfuron (98.5%) were obtained from Sigma–Aldrich (St. Louis, MO, USA). The individual stock standard solution was prepared in methanol at a concentration of 100 g/mL and stored at 4⬚C. The working standard solutions were daily prepared by dilution of the stock standard solution with deionized water to the required concentrations. www.jss-journal.com
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Florisil (60–100 mesh) and primary secondary amine (PSA, particle size 40–60 m) were obtained from Agela Technologies (Beijing, China). Silica gel (100–150 mesh), aluminaB (100–200 mesh), and CN-silica (particle size 10 m) were obtained from Sinopharm Chemical Reagent (Shanghai, China). HPLC-grade acetonitrile and methanol were obtained from TEDIA Company (Fair Lawn, NJ, USA). Acetone, ethanol, ethyl acetate, dichloromethane, chlorobenzene, trichloromethane, carbon tetrachloride, dichloroethane, acetic acid, sodium chloride, and anhydrous sodium sulfate were all of analytical reagent grade or better and obtained from Sinopharm Chemical Reagent (Shanghai, China). Ultra high purity water from a Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA) was used in this work. Multiwalled carbon nanotubes (MWNTs) were kindly provided by the Institute of Nanometer Material, Central China Normal University. The method of preparation and characteristics of the MWNTs was reported previously [32]. Before use, MWNTs were refluxed with concentrated nitric acid for 1 h, and then washed with doubly distilled water until neutral pH was reached. The MWNTs were dried at 100⬚C for 2 h and then stored in a desiccator for further use. Various species of tea samples, including green, black, and Oolong teas were purchased from the market of Wuhan (Hubei, China). Tea samples were chopped and homogenized, and then placed in a plastic zipper bag and stored at 4⬚C until analysis. Spiked tea samples for extraction studies and recovery determination were prepared by adding an appropriate amount of a working standard solution to 0.5 g of blank tea sample, which was left to stand for 30 min to allow the spiked solution to impregnate the tea. A residue-free green tea was used as a control sample for method optimization and validation.
2.2 Instrumentation The chromatographic analysis was performed on an Agilent 1100 HPLC system equipped with a manual injector and a diode array detector. A personal computer equipped with an Agilent ChemStation program for LC was used to process chromatographic data. A ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm id, 5 m) was used for separation. Optimum separation was achieved with a binary mobile phase that consisted of methanol and 0.05% acetic acid solution (50:50, v/v), with a flow rate of 1.2 mL/min. The column temperature was set at 30⬚C and the diode array detector monitoring wavelength was set at 240 nm. An 80–2 centrifuge (Changzhou Guohua Electric Appliance, Changzhou, China) was used for phase separation.
2.3 MSPD–DLLME procedure An aliquot of tea sample (accurately 20 mg) was placed in a glass mortar, and was blended with 60 mg of CN-silica (dis C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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persant) and 20 mg of anhydrous sodium sulfate for 5 min using a pestle. The homogeneous mixture was transferred into a 1 mL syringe with a frit containing a layer of 50 mg of anhydrous sodium sulfate and another layer of 5 mg carbon nanotubes packing at the bottom. A second frit was placed over the homogenized sample and slightly compressed with a plunger in order to avoid undesirable channels. The cartridge was first rinsed with 3 mL of n-hexane, and then the target analytes retained on the adsorbent were eluted with 2.5 mL of dichloromethane (containing 5 L of acetic acid). The eluent was collected into a test tube, evaporated to dryness under a gentle flow of nitrogen, and then redissolved with 0.5 mL methanol (as disperser solvent in DLLME) containing 40 L chlorobenzene (as extraction solvent). The resultant solution was rapidly injected into 5.0 mL deionized water (containing 1% w/v NaCl and the pH was adjusted to 2.0 with HCl) in a 10 mL screw-cap glass test tube with a conical bottom by 1.00 mL syringe. After vortexing for 15 s, a cloudy solution that consists of fine droplets of chlorobenzene dispersed into aqueous sample was formed, and the analytes were extracted into the fine droplets. After centrifuging for 5 min at 3000 rpm, the extraction solvent was sedimented in the bottom of the conical test tube. The sedimented phase was transferred into a small vial by 50 L syringe, evaporated to dryness under a gentle flow of nitrogen, and then accurately reconstituted with 50 L of methanol. After filtration with a 0.45 m membrane, 20 L of the filtrate was injected into HPLC for analysis.
3 Results and discussion 3.1 Optimization of MSPD procedure 3.1.1 Selection of dispersant and ratio of dispersant to sample The sorbents used in MSPD are not only for the adsorption and separation of target compounds, but also a solid blending support to disrupt and disperse the sample. Selection of a suitable sorbent is determined by the polarity of the analytes and the nature of the matrix. Several polar adsorbents, including silica gel, florisil, CN-silica, alumina-B, and PSA were investigated as the dispersant in this work, and the results are shown in Fig. 2. When silica gel was used as dispersant, some interfering peaks appeared and overlapped with the peak of target compounds. The highest extraction yield was obtained with CN-silica mainly because of its strong polarity. Thus, CN-silica was selected as dispersant in this work. A suitable ratio of dispersant to sample could increase the interface area between the analytes and sorbent, and allow complete adsorption of the sample components and to facilitate the transfer into the cartridge. Four different ratios of CN-silica to sample mass, 1:1, 2:1, 3:1, and 4:1, were evaluated, and the results showed that the highest extraction yield was achieved at the ratio of 3:1. Thereby, 3:1 was adopted www.jss-journal.com
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Figure 2. Effect of different dispersants on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
Figure 3. Effect of different elution solvents on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of elution solvent. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
as the optimized ratio of dispersant to sample for further experiments.
3.1.2 Selection of elution solvent and volume The selection of an appropriate eluting solvent was also important since a suitable solvent leads to the selective elution of the target analytes from the MSPD cartridge. After washing the cartridge with n-hexane, four different organic solvents including ethyl acetate, dichloromethane, acetone, acetonitrile were investigated as eluting solvent in this work. It is
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seen from Fig. 3 that the highest extraction yield of the target compounds was obtained with using dichloromethane as the elution solvent. So, dichloromethane was selected as the elution solvent for further experiments. For the purpose of using the minimum volume of eluting solvent to efficiently elute the analytes, different volumes of dichloromethane (1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 mL) were investigated. The results revealed that the extraction yields of the analytes increased with the increase in dichloromethane volume from 1.0 to 2.5 mL, and then retained constant even further increasing the volume to 4.0 mL. Thus, 2.5 mL of dichloromethane was used as the elution solvent.
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Sulfonylureas are weak acids and only achieve an overall neutral state in aqueous solution at pH values below pKa [33]. Increasing the acidity of the elution solvent should be beneficial to the elution of the analytes. Elution solvents with different volume of acetic acid (0–20 L) were tested in this work. The experimental results indicated that the extraction yields of SUHs increased when the volume of acetic acid in the elution solvent increased from 0 to 5 L, and then kept constant with the further increasing of the volume. Therefore, elution solvent with 5 L of acetic acid was used in this work.
3.1.3 Selection of cleanup sorbent When eluting of target compounds from the MSPD cartridge, the matrix components such as pigments, polyphenols, alkaloids, amino acids, and other polar substances in tea may be coeluted and interfere with the analysis of target compounds. Cleanup sorbent was needed to remove impurities in the eluent. Activated carbon and carbon nanotubes that are widely used as sorbent for the sorption of environmental contaminants were tested as the cleanup sorbents [34, 35]. The sorbents were separately packed at the bottom of the MSPD cartridge. When the eluent passed through the MSPD cartridge, the impurities could be adsorbed onto the sorbent and the analytes passed through the column. The results indicated that activated carbon has the stronger ability of adsorption pigments than carbon nanotubes, but it can also adsorb target compounds resulting in lower extraction yield. Carbon nanotubes could retain most of the impurities and get a higher extraction yield than activated carbon because they are oxidized to some extent and do not have the capability to absorb the neutral pesticides. Therefore, carbon nanotubes were chosen as the cleanup sorbent in the present study, and 5 mg carbon nanotubes was sufficient to remove the impurities in the eluent.
3.2 Optimization of DLLME procedure
Figure 4. Effect of the volume of extraction solvent on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; C6 H5 Cl as extraction solvent.
in water. Therefore, C6 H5 Cl was selected as the extraction solvent in the subsequent experiment. In order to examine the effect of the extraction solvent volume, 0.50 mL methanol containing different volumes of C6 H5 Cl (35, 40, 50, 60, and 70 L) were subjected to the DLLME procedure. Figure 4 shows the variation of the extraction yield versus the volume of the extraction solvent. As can be seen, the extraction yields of SUHs increased with increasing the volume of the extraction solvent from 35 to 40 L, and then remained almost constant with further increasing the volume over 40 L. Thus 40 L of C6 H5 Cl was chosen as the optimum volume of the extraction solvent. 3.2.2 Selection of dispersive solvent and its volume
3.2.1 Selection of extraction solvent and its volume The type of extraction solvent used in DLLME is an essential consideration for efficient extraction. The extraction solvent should have a high density, low solubility in water, high extraction capability for the interested compounds and form a stable two-phase system in the presence of the dispersive solvent when injected into an aqueous solution [36,37]. Based on these considerations, C6 H5 Cl, CCl4 , CHCl3 , and C2 H6 Cl2 were evaluated as extraction solvent by using 0.50 mL methanol as dispersive solvent. In the case of CHCl3 and C2 H6 Cl2 tested as extraction solvent, two-phase system was not observed. With C6 H5 Cl and CCl4 as extraction solvent, the stable two-phase system could form and the higher extraction yield was obtained with using C6 H5 Cl as extraction solvent because of its stronger polarity and lower solubility C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
For the DLLME method, the dispersive solvent should be miscible with both water and the extraction solvent. With C6 H5 Cl as extraction solvent, the use of acetonitrile, acetone, ethanol, and methanol as dispersive solvent could produce a two-phase system. The effect of these solvents on the extraction yield was investigated using 0.5 mL of each solvent containing 40 L of C6 H5 Cl subjected to the DLLME procedure, and the results are shown in Fig. 5. As can be seen, methanol gave the highest extraction yield among the four solvents studied, so methanol was selected as dispersive solvent in the DLLME procedure. In order to obtain optimized volume of dispersive solvent, various experiments were performed by using different volumes of methanol (0.25, 0.50, 0.75, 1.00, and 1.25 mL) containing 40 L C6 H5 Cl. The result showed that the extraction www.jss-journal.com
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Figure 5. Effect of different dispersive solvents on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
yield increased with the increase of the volume of methanol to 0.50 mL. Reduction in the extraction yield was observed after the volume of methanol exceeded 0.50 mL. At low volume, methanol could not disperse C6 H5 Cl properly. Conversely, at high volume, the solubility of SUHs in aqueous solution increased by the increase of the volume of methanol. Thereby, 0.50 mL methanol was chosen as the optimum volume.
the aqueous phase increases, and the extraction solvent could not be dispersed properly. Based on the experimental results, 1% NaCl was added in the aqueous phase in the DLLME procedure.
3.3 Method validation 3.2.3 Effect of pH of the aqueous phase Because the SUHs are weakly acidic compounds with pKa values from 3.6 to 5.2, the aqueous solution should be acidic to effectively deionize the SUHs and consequently reduce their solubility within the aqueous solution. The effect of pH of the aqueous phase on the extraction yield was studied in the range of 1.0–6.0. The results indicated that the highest extraction yield was obtained at pH 2.0. Therefore, the aqueous phase was adjusted to pH 2.0 with HCl prior to the DLLME procedure. 3.2.4 Effect of salt addition The effect of salt addition in the aqueous phase on the extraction yield was investigated by adding different amounts of NaCl (0–6%, w/v). The results showed that the extraction yield increased with the increasing of NaCl concentration to 1%. At higher concentration, a decrease in the extraction yield was observed. For the DLLME process, the effect of salt addition can be considered as the result of two major competitive effects: salting-out effect and viscous resistance effect [38]. The salting-out effect can decrease the solubility of analytes in the aqueous phase and promote the transfer of the analytes toward the organic phase, thus improve the extraction yield. However, at high salt concentration, the viscous resistance of
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The target compounds were identified by comparing their retention times with those of the authentic standard analytes. Chromatograms obtained for green tea samples (unspiked and spiked with four SUHs at two levels) after the MSPD– DLLME procedure under the optimum conditions are shown in Fig. 6, and no interferences originating from the tea matrices were observed.
3.3.1 Method performance The analytical performance of the proposed MSPD–DLLME– HPLC method for the determination of four SUHs was evaluated using the spiked tea samples under the optimal conditions, and the detailed results are shown in Table 1. Calibration curves were constructed using the areas of the chromatographic peaks measured at six increasing spiked concentrations ranging from 5.0 to 10000 ng/g. In the selected range 5–10000 ng/g, a good correlation coefficient (r2 ) could be obtained for all analytes ranging from 0.9959 to 0.9998. The repeatability study was carried out by seven parallel experiments at the spiked concentration of 50 ng/g for each of the SUHs, and the RSDs varied from 4.3–6.8%. The LODs, calculated as the analyte lowest concentrations that yield a S/N of 3, were in a range of 1.31–2.81 ng/g.
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Figure 6. Chromatograms obtained for green tea samples after MSPD-DLLME procedure under the optimal conditions. (A) Green tea; (B) green tea fortified with 10 ng/g sulfonylurea herbicides; (C) green tea fortified with 50 ng/g sulfonylurea herbicides. Peak identification: 1Chlorsulfuron, 2-Bensulfuron-methyl, 3Chlorimuron-ethyl, 4-Pyrazosulfuron. Table 1. Analytical performance data of the MSPD–DLLME method
Compound
Calibration curve
Linear range (ng/g)
r2
LOD (ng/g
RSD (%)a)
Chlorsulfuron Bensulfuron-methyl Chlorimuron-ethyl Pyrazosulfuron
Y = 2819.8X + 40.8 Y = 2530.3X + 125.1 Y = 2411.1X + 105.6 Y = 3549.3X + 79.6
10–10 000 5–10 000 10–10 000 10–10 000
0.9984 0.9978 0.9959 0.9998
2.81 1.31 2.45 2.32
6.3 5.4 6.8 4.3
a) Seven parallel experiments at the spiked concentration of 50 ng/g. Table 2. Analytical results of SUHs residues and recoveries in tea samples (n = 5)
Compound
Spiked (ng/g)
Green tea Found (ng/g)
Chlorsulfuron
Bensulfuron-methyl
Chlorimuron-ethyl
Pyrazosulfuron
0 25 50 0 25 50 0 25 50 0 25 50
nd 23.7 49.6 nd 21.3 50.3 nd 20.4 46.0 nd 22.1 48.4
Black tea Recovery (%)
RSD (%)
94.8 99.2
6.54 3.68
85.2 100.6
6.68 3.12
81.6 92.0
6.89 4.38
88.6 96.8
6.12 2.85
3.3.2 Real samples analysis To evaluate the applicability of the proposed method in real samples, three kinds of tea samples (green, black, and Oolong teas) from the local supermarket were analyzed using the proposed method. The results showed that the tea samples
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Found (ng/g) nd 21.0 54.6 nd 19.7 53.5 nd 18.9 50.6 nd 19.5 55.3
Oolong tea Recovery (%)
RSD (%)
84.2 109.2
5.65 3.19
78.8 107.0
5.12 3.81
75.6 101.2
6.76 4.49
78.0 110.6
5.42 3.42
Found (ng/g) nd 21.8 49.1 nd 18.2 48.3 nd 19.2 52.1 nd 21.3 51.8
Recovery (%)
RSD (%)
87.2 98.2
4.76 4.25
72.8 96.6
6.95 4.37
76.8 104.2
6.35 4.18
85.2 103.6
5.37 3.78
were free of the four SUHs. The recovery experiments were carried out to evaluate the accuracy of the method. The tea samples spiked with four SUHs at 25 and 50 ng/g levels were extracted under the optimized conditions for five times at each level, and the average recoveries are shown in Table 2. The overall recoveries were in the range of 72.8–110.6%, with
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the RSDs less than 6.95%. These results demonstrated that the established method was reliable for the determination of SUHs in tea.
4 Conclusions In this study, MSPD combined with DLLME was developed as a new sample pretreatment approach for the reliable determination of some sulfonylurea herbicides in tea. The proposed MSPD–DLLME method combines the good clarification of MSPD for complex samples and the high enrichment capability of DLLME. The present method had many practical advantages compared with traditional techniques, including simplicity and efficiency of the extraction method, consuming less sample and organic solvent, high sensitivity, and rapidity. The results indicated that the proposed method is an efficient sample pretreatment procedure for analyzing SUHs in complex matrix samples.
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[33] Niu, H. Y., Shi, Y. L., Cai, Y. Q., Wei, F. S., Jiang, G. B., Microchim. Acta 2009, 164, 431–438. [34] Mauter, M. S., Elimelech, M., Environ. Sci. Technol. 2008, 42, 5843–5859. [35] Yang, K., Xing, B. S., Chem. Rev. 2010, 110, 5989–6008.
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Research Article
Determination of four sulfonylurea herbicides in tea by matrix solid-phase dispersion cleanup followed by dispersive liquid–liquid microextraction Matrix solid-phase dispersion combined with dispersive liquid–liquid microextraction has been developed as a new sample pretreatment method for the determination of four sulfonylurea herbicides (chlorsulfuron, bensulfuron-methyl, chlorimuron-ethyl, and pyrazosulfuron) in tea by high-performance liquid chromatography with diode array detection. The extraction and cleanup by matrix solid-phase dispersion was carried out by using CN-silica as dispersant and carbon nanotubes as cleanup sorbent eluted with acidified dichloromethane. The eluent of matrix solid-phase dispersion was evaporated and redissolved in 0.5 mL methanol, and used as the dispersive solvent of the following dispersive liquid–liquid microextraction procedure for further purification and enrichment of the target analytes before high-performance liquid chromatography analysis. Under the optimum conditions, the method yielded a linear calibration curve in the concentration range from 5.0 to 10 000 ng/g for target analytes with a correlation coefficients (r2 ) ranging from 0.9959 to 0.9998. The limits of detection for the analytes were in the range of 1.31–2.81 ng/g. Recoveries of the four sulfonylurea herbicides at two fortification levels were between 72.8 and 110.6% with relative standard deviations lower than 6.95%. The method was successfully applied to the analysis of four sulfonylurea herbicides in several tea samples. Keywords: Dispersive liquid–liquid microextraction / High-performance liquid chromatography / Matrix solid-phase dispersion / Sulfonylurea herbicides / Tea samples DOI 10.1002/jssc.201400449
1 Introduction Tea is one of the most consumed nonalcoholic beverages worldwide and valued for its specific aroma and flavor as well as potential health-promoting properties [1]. China is one of the largest tea-producing and exporting countries in the world. To control pest and disease, pesticides are widely used in nearly every period of cultivation, storage, and product manufacturing processes of tea. Pesticides residue may be present in tea and cause health threats to human through tea consumption [2]. In order to protect the health of consumers and regulate the international tea trade, several countries, and international organizations have defined maximum residue limits of pesticides for tea [3]. As the maximum residue limits have become stricter and comprehensive, the development Correspondence: Dr. Liang, Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Guizhishan, Wuhan, HuBei 430079, China E-mail: [email protected]
Abbreviations: DLLME, dispersive liquid–liquid microextraction; MSPD, matrix solid-phase dispersion; MWNT, multiwalled carbon nanotube; SUH, sulfonylurea herbicide
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of sensitive, accurate, and rapid methods for the analysis of pesticides in tea has become an important and urgent issue. The tea samples consist of complex matrix components including pigments, alkaloids and polyphenols, and lipophilic inclusions [4]. During the extraction process of pesticides residue analysis, they could be extracted with organic solvent that may lead to serious difficulties in qualitative and quantitative analysis of the analytes. In order to ensure the accuracy and reliability of experimental results, a sample pretreatment is often required not only to isolate and concentrate the target analytes, but also to reduce or eliminate the huge matrix effects caused by coextractives in tea extracts. The conventional methods of sample preparation for pesticides in tea include ultrasonic extraction, gel permeation chromatography, mechanical shaking extraction, and SPE. These techniques are easy to carry out and provide good recoveries. Nevertheless, they are time consuming, labor intensive, consume a high amount of organic solvent and have poor repeatability [5]. Dispersive liquid–liquid microextraction (DLLME) is a novel sample pretreatment method based on a ternary component solvent system [6]. In this method, water-immiscible extraction solvent dissolved in a water-miscible dispersive solvent is rapidly injected into an aqueous solution by syringe, and a cloudy solution is formed. The analyte in the sample is
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Figure 1. Chemical structure and pKa values of the studied sulfonylurea herbicides.
extracted into the fine droplets of extraction solvent. After extraction, phase separation is performed by centrifugation, and the enriched analyte in the sediment phase is determined by chromatography or spectrometry methods. The advantages of the DLLME method are simplicity of operation, rapidity, low organic solvent consumption, high recovery, and enrichment factor [7]. This method has been applied to determine a variety of organic pollutants and metal ions in environmental samples [8–11]. However, due to its lack of selectivity, DLLME is confronted with difficulty of extracting target analytes from complex matrix samples [12]. This is the main reason that the most reported applications of DLLME have been focused on simple matrix samples. Therefore, the exploration of the applications of DLLME technique for more complex matrix samples is desirable. The combination of DLLME with a sample cleanup method, such as SPE, dispersive SPE, and microwave-assisted extraction, has been reported [13–15]. One of the advantages of such a combination is that it can be used for complex matrix samples. Matrix solid-phase dispersion (MSPD) was introduced in 1989 by Barker for conducting simultaneous disruption and extraction of solid, semisolid, and highly viscous samples [16]. This method enables the simultaneous accomplishment of both extraction and cleanup steps, and has been demonstrated to be an attractive alternative to sample preparation of complex matrices. MSPD is achieved mainly through the dissolution and dispersion of the organic phase bound to the sorbent instead of solvent extraction, and thus it consumes far less organic solvent and requires a shorter extraction time compared with conventional extraction methods [17]. The application of MSPD has shown satisfactory results in the extraction of pesticides, antibiotics, additives, banned dyes, and other pollutants from a wide variety of complex samples [18–22]. To our knowledge, there are limited papers in the literature reporting the combination of MSPD with DLLME [23–25]. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The aim of this work was to develop a new sample pretreatment method by combining MSPD with DLLME, thus to enhance the selectivity of the DLLME method. In this study, the applicability of the proposed method was explored for the extraction of some sulfonylurea herbicides (SUHs) in tea samples prior to their analysis by HPLC. SUHs are used worldwide to control several broad-leaved weeds and some grasses in a variety of crops because of their low application rates, unprecedented herbicidal activity, and low mammalian toxicity [26, 27]. The mode of action of SUHs is inhibition of the biosynthesis of branched chain amino acids [28], and SUHs residues could stop cell division in plants and lead to yield reduction in sensitive crops [29, 30]. The maximum residual limit of SUHs in tea (0.05 mg/kg) was established by the European Union [31]. Therefore, efficient, reliable and sensitive analytical methods are indispensable for the control of SUHs residues. Four of the most widely used SUHs (chlorsulfuron, bensulfuron-methyl, chlorimuron-ethyl, and pyrazosulfuron, the structures of these molecules are shown in (Fig. 1)) in the local area were chosen as target analytical compounds. The factors affecting the detection sensitivity of the method are investigated in detail.
2 Materials and methods 2.1 Standards, reagents, and samples Chlorsulfuron (99.5%), Bensulfuron-methyl (99.0%), Chlorimuron-ethyl (99.0%), and Pyrazosulfuron (98.5%) were obtained from Sigma–Aldrich (St. Louis, MO, USA). The individual stock standard solution was prepared in methanol at a concentration of 100 g/mL and stored at 4⬚C. The working standard solutions were daily prepared by dilution of the stock standard solution with deionized water to the required concentrations. www.jss-journal.com
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Florisil (60–100 mesh) and primary secondary amine (PSA, particle size 40–60 m) were obtained from Agela Technologies (Beijing, China). Silica gel (100–150 mesh), aluminaB (100–200 mesh), and CN-silica (particle size 10 m) were obtained from Sinopharm Chemical Reagent (Shanghai, China). HPLC-grade acetonitrile and methanol were obtained from TEDIA Company (Fair Lawn, NJ, USA). Acetone, ethanol, ethyl acetate, dichloromethane, chlorobenzene, trichloromethane, carbon tetrachloride, dichloroethane, acetic acid, sodium chloride, and anhydrous sodium sulfate were all of analytical reagent grade or better and obtained from Sinopharm Chemical Reagent (Shanghai, China). Ultra high purity water from a Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA) was used in this work. Multiwalled carbon nanotubes (MWNTs) were kindly provided by the Institute of Nanometer Material, Central China Normal University. The method of preparation and characteristics of the MWNTs was reported previously [32]. Before use, MWNTs were refluxed with concentrated nitric acid for 1 h, and then washed with doubly distilled water until neutral pH was reached. The MWNTs were dried at 100⬚C for 2 h and then stored in a desiccator for further use. Various species of tea samples, including green, black, and Oolong teas were purchased from the market of Wuhan (Hubei, China). Tea samples were chopped and homogenized, and then placed in a plastic zipper bag and stored at 4⬚C until analysis. Spiked tea samples for extraction studies and recovery determination were prepared by adding an appropriate amount of a working standard solution to 0.5 g of blank tea sample, which was left to stand for 30 min to allow the spiked solution to impregnate the tea. A residue-free green tea was used as a control sample for method optimization and validation.
2.2 Instrumentation The chromatographic analysis was performed on an Agilent 1100 HPLC system equipped with a manual injector and a diode array detector. A personal computer equipped with an Agilent ChemStation program for LC was used to process chromatographic data. A ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm id, 5 m) was used for separation. Optimum separation was achieved with a binary mobile phase that consisted of methanol and 0.05% acetic acid solution (50:50, v/v), with a flow rate of 1.2 mL/min. The column temperature was set at 30⬚C and the diode array detector monitoring wavelength was set at 240 nm. An 80–2 centrifuge (Changzhou Guohua Electric Appliance, Changzhou, China) was used for phase separation.
2.3 MSPD–DLLME procedure An aliquot of tea sample (accurately 20 mg) was placed in a glass mortar, and was blended with 60 mg of CN-silica (dis C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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persant) and 20 mg of anhydrous sodium sulfate for 5 min using a pestle. The homogeneous mixture was transferred into a 1 mL syringe with a frit containing a layer of 50 mg of anhydrous sodium sulfate and another layer of 5 mg carbon nanotubes packing at the bottom. A second frit was placed over the homogenized sample and slightly compressed with a plunger in order to avoid undesirable channels. The cartridge was first rinsed with 3 mL of n-hexane, and then the target analytes retained on the adsorbent were eluted with 2.5 mL of dichloromethane (containing 5 L of acetic acid). The eluent was collected into a test tube, evaporated to dryness under a gentle flow of nitrogen, and then redissolved with 0.5 mL methanol (as disperser solvent in DLLME) containing 40 L chlorobenzene (as extraction solvent). The resultant solution was rapidly injected into 5.0 mL deionized water (containing 1% w/v NaCl and the pH was adjusted to 2.0 with HCl) in a 10 mL screw-cap glass test tube with a conical bottom by 1.00 mL syringe. After vortexing for 15 s, a cloudy solution that consists of fine droplets of chlorobenzene dispersed into aqueous sample was formed, and the analytes were extracted into the fine droplets. After centrifuging for 5 min at 3000 rpm, the extraction solvent was sedimented in the bottom of the conical test tube. The sedimented phase was transferred into a small vial by 50 L syringe, evaporated to dryness under a gentle flow of nitrogen, and then accurately reconstituted with 50 L of methanol. After filtration with a 0.45 m membrane, 20 L of the filtrate was injected into HPLC for analysis.
3 Results and discussion 3.1 Optimization of MSPD procedure 3.1.1 Selection of dispersant and ratio of dispersant to sample The sorbents used in MSPD are not only for the adsorption and separation of target compounds, but also a solid blending support to disrupt and disperse the sample. Selection of a suitable sorbent is determined by the polarity of the analytes and the nature of the matrix. Several polar adsorbents, including silica gel, florisil, CN-silica, alumina-B, and PSA were investigated as the dispersant in this work, and the results are shown in Fig. 2. When silica gel was used as dispersant, some interfering peaks appeared and overlapped with the peak of target compounds. The highest extraction yield was obtained with CN-silica mainly because of its strong polarity. Thus, CN-silica was selected as dispersant in this work. A suitable ratio of dispersant to sample could increase the interface area between the analytes and sorbent, and allow complete adsorption of the sample components and to facilitate the transfer into the cartridge. Four different ratios of CN-silica to sample mass, 1:1, 2:1, 3:1, and 4:1, were evaluated, and the results showed that the highest extraction yield was achieved at the ratio of 3:1. Thereby, 3:1 was adopted www.jss-journal.com
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Figure 2. Effect of different dispersants on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
Figure 3. Effect of different elution solvents on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of elution solvent. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
as the optimized ratio of dispersant to sample for further experiments.
3.1.2 Selection of elution solvent and volume The selection of an appropriate eluting solvent was also important since a suitable solvent leads to the selective elution of the target analytes from the MSPD cartridge. After washing the cartridge with n-hexane, four different organic solvents including ethyl acetate, dichloromethane, acetone, acetonitrile were investigated as eluting solvent in this work. It is
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seen from Fig. 3 that the highest extraction yield of the target compounds was obtained with using dichloromethane as the elution solvent. So, dichloromethane was selected as the elution solvent for further experiments. For the purpose of using the minimum volume of eluting solvent to efficiently elute the analytes, different volumes of dichloromethane (1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 mL) were investigated. The results revealed that the extraction yields of the analytes increased with the increase in dichloromethane volume from 1.0 to 2.5 mL, and then retained constant even further increasing the volume to 4.0 mL. Thus, 2.5 mL of dichloromethane was used as the elution solvent.
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Sulfonylureas are weak acids and only achieve an overall neutral state in aqueous solution at pH values below pKa [33]. Increasing the acidity of the elution solvent should be beneficial to the elution of the analytes. Elution solvents with different volume of acetic acid (0–20 L) were tested in this work. The experimental results indicated that the extraction yields of SUHs increased when the volume of acetic acid in the elution solvent increased from 0 to 5 L, and then kept constant with the further increasing of the volume. Therefore, elution solvent with 5 L of acetic acid was used in this work.
3.1.3 Selection of cleanup sorbent When eluting of target compounds from the MSPD cartridge, the matrix components such as pigments, polyphenols, alkaloids, amino acids, and other polar substances in tea may be coeluted and interfere with the analysis of target compounds. Cleanup sorbent was needed to remove impurities in the eluent. Activated carbon and carbon nanotubes that are widely used as sorbent for the sorption of environmental contaminants were tested as the cleanup sorbents [34, 35]. The sorbents were separately packed at the bottom of the MSPD cartridge. When the eluent passed through the MSPD cartridge, the impurities could be adsorbed onto the sorbent and the analytes passed through the column. The results indicated that activated carbon has the stronger ability of adsorption pigments than carbon nanotubes, but it can also adsorb target compounds resulting in lower extraction yield. Carbon nanotubes could retain most of the impurities and get a higher extraction yield than activated carbon because they are oxidized to some extent and do not have the capability to absorb the neutral pesticides. Therefore, carbon nanotubes were chosen as the cleanup sorbent in the present study, and 5 mg carbon nanotubes was sufficient to remove the impurities in the eluent.
3.2 Optimization of DLLME procedure
Figure 4. Effect of the volume of extraction solvent on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent (methanol) volume, 0.5 mL; C6 H5 Cl as extraction solvent.
in water. Therefore, C6 H5 Cl was selected as the extraction solvent in the subsequent experiment. In order to examine the effect of the extraction solvent volume, 0.50 mL methanol containing different volumes of C6 H5 Cl (35, 40, 50, 60, and 70 L) were subjected to the DLLME procedure. Figure 4 shows the variation of the extraction yield versus the volume of the extraction solvent. As can be seen, the extraction yields of SUHs increased with increasing the volume of the extraction solvent from 35 to 40 L, and then remained almost constant with further increasing the volume over 40 L. Thus 40 L of C6 H5 Cl was chosen as the optimum volume of the extraction solvent. 3.2.2 Selection of dispersive solvent and its volume
3.2.1 Selection of extraction solvent and its volume The type of extraction solvent used in DLLME is an essential consideration for efficient extraction. The extraction solvent should have a high density, low solubility in water, high extraction capability for the interested compounds and form a stable two-phase system in the presence of the dispersive solvent when injected into an aqueous solution [36,37]. Based on these considerations, C6 H5 Cl, CCl4 , CHCl3 , and C2 H6 Cl2 were evaluated as extraction solvent by using 0.50 mL methanol as dispersive solvent. In the case of CHCl3 and C2 H6 Cl2 tested as extraction solvent, two-phase system was not observed. With C6 H5 Cl and CCl4 as extraction solvent, the stable two-phase system could form and the higher extraction yield was obtained with using C6 H5 Cl as extraction solvent because of its stronger polarity and lower solubility C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
For the DLLME method, the dispersive solvent should be miscible with both water and the extraction solvent. With C6 H5 Cl as extraction solvent, the use of acetonitrile, acetone, ethanol, and methanol as dispersive solvent could produce a two-phase system. The effect of these solvents on the extraction yield was investigated using 0.5 mL of each solvent containing 40 L of C6 H5 Cl subjected to the DLLME procedure, and the results are shown in Fig. 5. As can be seen, methanol gave the highest extraction yield among the four solvents studied, so methanol was selected as dispersive solvent in the DLLME procedure. In order to obtain optimized volume of dispersive solvent, various experiments were performed by using different volumes of methanol (0.25, 0.50, 0.75, 1.00, and 1.25 mL) containing 40 L C6 H5 Cl. The result showed that the extraction www.jss-journal.com
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Figure 5. Effect of different dispersive solvents on the extraction yields of sulfonylurea herbicides. MSPD conditions: 0.02 g tea sample spiked with 0.5 mg/kg SUHs, 0.06 g of CN-silica as dispersant, 5 mg of carbon nanotubes as cleanup sorbent, washed with 3 mL of n-hexane and eluted with 2.5 mL of dichloromethane. DLLME conditions: 5.0 mL deionized water (pH 2.0 and containing 1% NaCl); dispersive solvent volume, 0.5 mL; extraction solvent (C6 H5 Cl) volume, 40 L.
yield increased with the increase of the volume of methanol to 0.50 mL. Reduction in the extraction yield was observed after the volume of methanol exceeded 0.50 mL. At low volume, methanol could not disperse C6 H5 Cl properly. Conversely, at high volume, the solubility of SUHs in aqueous solution increased by the increase of the volume of methanol. Thereby, 0.50 mL methanol was chosen as the optimum volume.
the aqueous phase increases, and the extraction solvent could not be dispersed properly. Based on the experimental results, 1% NaCl was added in the aqueous phase in the DLLME procedure.
3.3 Method validation 3.2.3 Effect of pH of the aqueous phase Because the SUHs are weakly acidic compounds with pKa values from 3.6 to 5.2, the aqueous solution should be acidic to effectively deionize the SUHs and consequently reduce their solubility within the aqueous solution. The effect of pH of the aqueous phase on the extraction yield was studied in the range of 1.0–6.0. The results indicated that the highest extraction yield was obtained at pH 2.0. Therefore, the aqueous phase was adjusted to pH 2.0 with HCl prior to the DLLME procedure. 3.2.4 Effect of salt addition The effect of salt addition in the aqueous phase on the extraction yield was investigated by adding different amounts of NaCl (0–6%, w/v). The results showed that the extraction yield increased with the increasing of NaCl concentration to 1%. At higher concentration, a decrease in the extraction yield was observed. For the DLLME process, the effect of salt addition can be considered as the result of two major competitive effects: salting-out effect and viscous resistance effect [38]. The salting-out effect can decrease the solubility of analytes in the aqueous phase and promote the transfer of the analytes toward the organic phase, thus improve the extraction yield. However, at high salt concentration, the viscous resistance of
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The target compounds were identified by comparing their retention times with those of the authentic standard analytes. Chromatograms obtained for green tea samples (unspiked and spiked with four SUHs at two levels) after the MSPD– DLLME procedure under the optimum conditions are shown in Fig. 6, and no interferences originating from the tea matrices were observed.
3.3.1 Method performance The analytical performance of the proposed MSPD–DLLME– HPLC method for the determination of four SUHs was evaluated using the spiked tea samples under the optimal conditions, and the detailed results are shown in Table 1. Calibration curves were constructed using the areas of the chromatographic peaks measured at six increasing spiked concentrations ranging from 5.0 to 10000 ng/g. In the selected range 5–10000 ng/g, a good correlation coefficient (r2 ) could be obtained for all analytes ranging from 0.9959 to 0.9998. The repeatability study was carried out by seven parallel experiments at the spiked concentration of 50 ng/g for each of the SUHs, and the RSDs varied from 4.3–6.8%. The LODs, calculated as the analyte lowest concentrations that yield a S/N of 3, were in a range of 1.31–2.81 ng/g.
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Figure 6. Chromatograms obtained for green tea samples after MSPD-DLLME procedure under the optimal conditions. (A) Green tea; (B) green tea fortified with 10 ng/g sulfonylurea herbicides; (C) green tea fortified with 50 ng/g sulfonylurea herbicides. Peak identification: 1Chlorsulfuron, 2-Bensulfuron-methyl, 3Chlorimuron-ethyl, 4-Pyrazosulfuron. Table 1. Analytical performance data of the MSPD–DLLME method
Compound
Calibration curve
Linear range (ng/g)
r2
LOD (ng/g
RSD (%)a)
Chlorsulfuron Bensulfuron-methyl Chlorimuron-ethyl Pyrazosulfuron
Y = 2819.8X + 40.8 Y = 2530.3X + 125.1 Y = 2411.1X + 105.6 Y = 3549.3X + 79.6
10–10 000 5–10 000 10–10 000 10–10 000
0.9984 0.9978 0.9959 0.9998
2.81 1.31 2.45 2.32
6.3 5.4 6.8 4.3
a) Seven parallel experiments at the spiked concentration of 50 ng/g. Table 2. Analytical results of SUHs residues and recoveries in tea samples (n = 5)
Compound
Spiked (ng/g)
Green tea Found (ng/g)
Chlorsulfuron
Bensulfuron-methyl
Chlorimuron-ethyl
Pyrazosulfuron
0 25 50 0 25 50 0 25 50 0 25 50
nd 23.7 49.6 nd 21.3 50.3 nd 20.4 46.0 nd 22.1 48.4
Black tea Recovery (%)
RSD (%)
94.8 99.2
6.54 3.68
85.2 100.6
6.68 3.12
81.6 92.0
6.89 4.38
88.6 96.8
6.12 2.85
3.3.2 Real samples analysis To evaluate the applicability of the proposed method in real samples, three kinds of tea samples (green, black, and Oolong teas) from the local supermarket were analyzed using the proposed method. The results showed that the tea samples
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Found (ng/g) nd 21.0 54.6 nd 19.7 53.5 nd 18.9 50.6 nd 19.5 55.3
Oolong tea Recovery (%)
RSD (%)
84.2 109.2
5.65 3.19
78.8 107.0
5.12 3.81
75.6 101.2
6.76 4.49
78.0 110.6
5.42 3.42
Found (ng/g) nd 21.8 49.1 nd 18.2 48.3 nd 19.2 52.1 nd 21.3 51.8
Recovery (%)
RSD (%)
87.2 98.2
4.76 4.25
72.8 96.6
6.95 4.37
76.8 104.2
6.35 4.18
85.2 103.6
5.37 3.78
were free of the four SUHs. The recovery experiments were carried out to evaluate the accuracy of the method. The tea samples spiked with four SUHs at 25 and 50 ng/g levels were extracted under the optimized conditions for five times at each level, and the average recoveries are shown in Table 2. The overall recoveries were in the range of 72.8–110.6%, with
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the RSDs less than 6.95%. These results demonstrated that the established method was reliable for the determination of SUHs in tea.
4 Conclusions In this study, MSPD combined with DLLME was developed as a new sample pretreatment approach for the reliable determination of some sulfonylurea herbicides in tea. The proposed MSPD–DLLME method combines the good clarification of MSPD for complex samples and the high enrichment capability of DLLME. The present method had many practical advantages compared with traditional techniques, including simplicity and efficiency of the extraction method, consuming less sample and organic solvent, high sensitivity, and rapidity. The results indicated that the proposed method is an efficient sample pretreatment procedure for analyzing SUHs in complex matrix samples.
5 References
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