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Validation of method for determination of different classes of pesticides in aqueous samples by dispersive liquid–liquid microextraction with liquid chromatography–tandem mass spectrometric detection
Analytica Chimica Acta 665 (2010) 55–62
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Validation of method for determination of different classes of pesticides in aqueous samples by dispersive liquid–liquid microextraction with liquid chromatography–tandem mass spectrometric detection Sergiane Souza Caldas, Fabiane Pinho Costa, Ednei Gilberto Primel ∗ Programa de Pós-Graduac¸ão em Química Tecnológica e Ambiental, Escola de Química e Alimentos, Universidade Federal do Rio Grande, Av Itália, km 8 s/n, Rio Grande, Rio Grande do Sul State, 96201-900, Brazil
a r t i c l e
i n f o
Article history: Received 27 November 2009 Received in revised form 2 March 2010 Accepted 6 March 2010 Available online 12 March 2010 Keywords: Dispersive liquid–liquid microextraction Pesticides Liquid chromatography–tandem mass spectrometry Sample preparation Aqueous samples
a b s t r a c t In this study, a simple, rapid and efficient method has been developed for the extraction and preconcentration of different classes of pesticides, carbofuran (insecticide), clomazone (herbicide) and tebuconazole (fungicide) in aqueous samples by dispersive liquid–liquid microextraction (DLLME) coupled with liquid chromatography–tandem mass spectrometric detection. Some experimental parameters that influence the extraction efficiency, such as the type and volume of the disperser solvents and extraction solvents, extraction time, speed of centrifugation, pH and addition of salt were examined and optimized. Under the optimum conditions, the recoveries of pesticides in water at spiking levels between 0.02 and 2.0 g L−1 ranged from 62.7% to 120.0%. The relative standard deviations varied between 1.9% and 9.1% (n = 3). The limits of quantification of the method considering a 50-fold preconcentration step were 0.02 g L−1 . The linearity of the method ranged from 1.0 to 1000 g L−1 for all compounds, with correlation coefficients varying from 0.9982 to 0.9992. Results show that the method we propose can meet the requirements for the determination of pesticides in water samples. The comparison of this method with solid-phase extraction indicates that DLLME is a simple, fast, and low-cost method for the determination of pesticides in natural waters. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Pesticides, including substances with high toxic effects and persistence in the environment, have been widely used in agriculture throughout the world. The pollution of environmental compartments involves a serious risk to the environment and to human health as well, due to either direct exposure or residues in food and drinking water [1,2]. Compounds belonging to different classes such as carbofuran (carbamate), clomazone (isoxazolidinone), and tebuconazole (triazole) have been widely used on a variety of crops in current agricultural practices worldwide; they have been found in natural waters in many regions of the world [3–6], and in Brazil [7–12]; therefore, it is important to have fast and simple methods to determine these compounds. Owing to the toxicity of pesticides, the US Environmental Protection Agency (EPA) and the European Union (EU) have included them in their list of priority pollutants [13]. The European Union (EU) establishes rigid limits for pesticides in water
∗ Corresponding author. Tel.: +55 53 32336956; fax: +55 53 32336961. E-mail addresses: [email protected], [email protected] (E.G. Primel). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.03.004
destined to human consumption, establishing 0.1 g L−1 for individual pesticides and 0.5 g L−1 for the sum of all pesticides [14]. Since the concentrations of these compounds are normally low (g L−1 or less), and due to the low detection levels required by regulatory bodies and the nature of the matrices in which the target compounds are present, efficient sample preparation and trace-level detection and identification are important aspects of analytical methods. New analytical methods that use less quantity of solvents and that are easy and rapid to execute are required for monitoring the widespread distribution of trace levels of pesticides [15,16]. Liquid–liquid extraction (LLE) is one of the oldest preconcentration and matrix isolation techniques in analytical chemistry [17], but it is still used in routine sample preparation for the determination of pesticides [18,19], not only because of its simplicity, robustness, minimal operator training, and efficiency, but also because of its wide acceptance in many standard methods [1]. However, conventional liquid–liquid extraction uses large amounts of solvent, which are often hazardous, and time-consuming to perform. Therefore, to overcome these disadvantages, techniques such as solid-phase extraction (SPE) have been developed. Solid-phase extraction is already a well-established and routine technique. SPE
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uses much less solvent than LLE, but can be relatively expensive [20]. Microextraction techniques, such as solid-phase microextraction (SPME), have been widely applied for the preconcentration and quantification of pesticides in water [21,22] and some of the potential advantages of microextraction techniques are miniaturization, reduction of organic solvent consumption and improvement in the selectivity of the extraction [23]. Solvent phase microextraction (single drop microextraction, liquid-phase microextraction, liquid–liquid microextraction, etc.), based on a traditional LLE technique, is a less known method that utilizes only a few microliters of organic solvent as the extracting phase [17,24]. Assadi and his co-workers have recently developed a new microextraction technique named dispersive liquid–liquid microextraction (DLLME) as a high-performance, rapid and inexpensive microextraction [20]. The basic principle of this method is the dispersion of extraction solvent (immiscible in water) assisted with disperser solvent (miscible in both water and extraction solvents) within an aqueous solution which generates a very high contact area between the aqueous phase and the extraction solvent. From a commercial, economic and environmental point of view, the advantages of the DLLME method over conventional solvent extraction methods are simplicity of operation, rapidity, low cost, easier manipulation, less amount of organic extraction solvents, high recovery and enrichment factor, and easier linkage to analytical methods [25], such as GC [15,26–28] and LC [29–32]. Its application has been extended to separation, preconcentration and determination of organic and inorganic compounds in liquid samples [15,23,33,34], but most studies of pesticides evaluated the extraction of compounds that belong to the same chemical group, with the same behavior [35,36]. An efficient technique for sample preparation should have the ability to extract compounds from different chemical classes in a single step. Therefore, the aim of this study is to examine the DLLME and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) suitability for the determination of carbofuran, clomazone and tebuconazole simultaneously in water samples. In addition, some important parameters, such as pH, kind and volume of extraction solvent and disperser solvent, extraction time, speed of centrifugation, and salt effect are investigated and optimized.
Other chemicals such as carbon tetrachloride, tetrachloroethylene, monochlorobenzene, 1,2-dichlorobenzene (spectroscopy grade) were purchased from Vetec (Rio de Janeiro, Brazil) and dichloromethane, chloroform (spectroscopy grade) and sodium chloride from Merck (Darmstadt, Germany). The LC-grade methanol, acetone and acetonitrile were bought from Mallinckrodt (Phillisburg, NJ, USA). Fresh tap water samples for the optimization of the method were collected in our laboratory. No filtration or any further treatment was conducted in tap water samples. For each batch of water samples, one blank water sample was analyzed.
2.2. Instruments Liquid chromatography with mass spectrometric detection was performed in a Waters Alliance 2695 Separations Module (Waters, Milford, USA) fitted with an autosampler, a membrane degasser and a quaternary pump. Mass spectrometry was performed on a Micromass Quattro Micro API (Waters, Milford, USA) with an ESI interface. The LC column was an XTerra 3.5 m particle size (50 × 3 mm i.d.) (Waters, Milford, MA, USA). Analytical instrument control, data acquisition, and treatment were performed by software Masslynx version 4.1 2005 (Micromass, Waters, Milford, MA, USA). A sample volume of 20 L was injected with an autosampler. The mobile phase was acetonitrile:water (52:48, v/v), acidified with 0.1% formic acid at a constant flow of 0.4 mL min−1 . Ionization of the compounds was made using electrospray interface (ESI) in the positive mode. The run time was 3 min. Typical interface conditions were optimized for maximum intensity of the precursor ions as follows: capillary voltage, 3.5 kV, nebulizer and desolvation (drying gas) flows were set at 350 and 150 L h−1 , respectively; source block and desolvation temperatures were 120 and 350 ◦ C, respectively. Nitrogen was used as nebulizing, desolvation and cone gas, and argon was used as collision gas. Optimization of the MS/MS conditions, i.e., choice of the ionization mode, identification of the parent and product ions, and selection of the cone and collision voltages, more favorable for the analysis of the target analytes, was performed with injection of their individual standard solutions. Both modes of ionization, negative and positive, were tested.
2. Experimental 2.1. Reagent and materials
2.3. Dispersive liquid–liquid microextraction procedure
Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7ylmethylcarbamate), clomazone (2-(2-chlorobenzyl)-4,4-dimethyl -1,2-oxazolidin-3-one) and tebuconazole ((RS)-1-p-chlorophenyl4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol) analytical standards (purity > 99%) were supplied by Sigma–Aldrich (São Paulo, Brazil) and phosphoric acid (85%) analytical grade, by Merck (Darmstadt, Germany). Water was purified with a Direct-Q UV3® (resistivity 18.2 M cm, Millipore, USA) water purification system (Millipore, Bedford, MA, USA). Individual pesticide stock solutions containing 1000 mg L−1 of the target compounds were prepared in methanol and stored at −18 ◦ C. Intermediate working standards mixtures in methanol, containing 100 mg L−1 for each pesticide, were prepared and used to prepare the working standard solutions containing 1.0, 0.1, 0.05, 0.01, 0.005, 0.001 mg L−1 ; these were used for spiking samples and for preparing the analytical curves. Working standard solutions were prepared monthly and were stored at −18 ◦ C, while the dilutions used for the analytical curves were prepared daily.
A 5.0 mL working standard solution (0.2 g L−1 ) was placed in a 10.0 mL glass tube with conical bottom. Acetonitrile (2.0 mL) as the disperser solvent, containing 60 L carbon tetrachloride as the extraction solvent, was rapidly injected into the sample solution by using a 5.0 mL syringe. In this step, a cloudy solution (water/acetonitrile/carbon tetrachloride) was formed in the test tube and the pesticides in the water sample were extracted into fine carbon tetrachloride droplets. The resultant cloudy solution was centrifuged for 5 min at 2000 rpm (Centribio 80-2B, Curitiba, PR, Brazil). After centrifuging, the dispersed fine droplets of carbon tetrachloride sedimented in the bottom of test tube (about 50 L). The volume of the sedimented phase was determined by a 100 L microsyringe. The sedimented phase was completely transferred to another tube using a 100 L LC syringe and, after evaporation of the solvent under nitrogen stream, the residue was redissolved in 100 L LC-grade methanol and injected into the separation system. All experiments were performed in triplicate and results are shown in plots and tables.
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2.4. Calculation of enrichment factor and extraction recovery The enrichment factor (EF) was defined as the ratio between the analyte concentration in the settled phase (Cset ) and the initial concentration of the analyte (Co ) in the aqueous sample [20,29]. EF =
Cset Co
(1)
The extraction recovery (R%) was defined as the percentage of the total analyte which was extracted in the settled phase. R% =
Cset × Vset × 100% Co × Vaq
(2)
where R%, Vsed , and Vaq are the extraction recovery, the volume of the sediment phase, and the volume of the aqueous sample, respectively [37]. 3. Results and discussion 3.1. Optimized dispersive liquid–liquid microextraction conditions In this study, DLLME combined with LC–ESI-MS/MS was used for preconcentration and determination of selected pesticides in aqueous samples. There are various parameters that affect the DLLME performance and efficiency, such as the kind and the volume of the extraction and the disperser solvents, the ionic strength, and the extraction time. These parameters were investigated and, then, the optimal conditions were selected. 3.1.1. Selection of the extraction solvent The choice of an appropriate extraction solvent plays the main role in this method in order to achieve good recovery, enrichment factor, and selectivity of the target compounds. The extraction solvent should demonstrate (a) good chromatographic behavior (it should not have elution strength higher than the mobile phase used in the separation system), (b) extraction capability of the interest compounds, (c) low solubility in water, and (d) should form a cloudy solution in the presence of a disperser solvent when injected into an aqueous solution (form very tiny droplets) [30,38]. The density of the extraction solvent is an important characteristic and should be known. The first DLLME work [20] and most studies that determine pesticide in waters uses solvents with higher density of water [32,39,40], but there is some works with modification of DLLME that uses solvents with lower density of water [41–43]. Among the solvents whose density is higher than water (mainly chlorinated solvents), monochlorobenzene (C6 H5 Cl) dichloromethane (CH2 Cl2 ), 1,2-dichlorobenzene (C6 H4 Cl2 ), chloroform (CHCl3 ), tetrachloroethylene (C2 Cl4 ) and carbon tetrachloride (CCl4 ) were examined in order to find the most suitable solvent for DLLME. In this study, combinations of 40 L of C2 Cl4 , CCl4 , CHCl3 , CH2 Cl2 , C6 H4 Cl2 and C6 H5 Cl with 1.5 mL of acetonitrile as disperser solvent were tested. Having CH2 Cl2 , C6 H5 Cl and CHCl3 as extraction solvents, a two-phase system was not observed with the disperser solvent when they were injected into 5.0 mL analytes solution in water. With CCl4 , C6 H4 Cl2 and C2 Cl4 as extraction solvents, a twophase system was formed and its sedimented phase could easily be removed with a microsyringe. The results revealed that CCl4 has the highest extraction efficiency (12.8–42.3%) in comparison with C6 H4 Cl2 (5.1–19.0%) and C2 Cl4 (3.0–21.4%) (Fig. 1). Probably, it happens because of the high density and water solubility among the other solvents that CCl4 shows (Table 1). Although the main characteristic of the extraction solvent is that it must show low solubility in water, because of the polar characteristics of the analytes, certain solubility in water
Fig. 1. Effect of different extraction solvents on the recovery of three pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL; extraction solvent, 40 L. Table 1 Physical properties of extraction solvents. Solvent
Density (g mL−1 )
Water solubility (g L−1 )
C6 H5 Cl C6 H4 Cl2 CH2 Cl2 CHCl3 CCl4 C2 Cl4
1.11 1.31 1.32 1.48 1.59 1.62
0.5 0.15 13.8 8.0 0.8 0.17
seems to have favored the extraction of compounds. Thereby, CCl4 was selected as the extraction solvent. 3.1.2. Selection of disperser solvent The miscibility of the disperser solvent in the organic phase (extraction solvent) and in the aqueous phase (sample solution) is the main point for the selection of the disperser solvent [20]. The disperser solvent should be miscible in water; it should also dissolve the extraction solvent. Acetone, acetonitrile and methanol, which have got this ability, were selected for this purpose. A series of sample solutions was studied, using 1.5 mL of each disperser solvent containing 40.0 L of extraction solvent (carbon tetrachloride). With CCl4 as extraction solvent, a two-phase system was formed with all three disperser solvents; in comparison with other disperser solvents, the recoveries for all analytes were higher using acetonitrile. In Fig. 2, recovery was plotted as a function of the type of disperser solvent. It can be concluded that acetonitrile has an advantage over the other solvents, showing more ability to cover the desired analytical spectrum in study than other solvents used. In other works, as performed by Fu et al. [39] and Liu et al. [44], acetonitrile was also the disperser solvent used for extraction of pesticides in water samples. For this reason, carbon tetrachloride and acetonitrile were chosen as extraction and disperser solvents, respectively, in the following studies.
Fig. 2. Effect of the disperser solvent on the recovery of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent volume, 1.5 mL; extraction solvent, 40.0 L CCl4 .
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Fig. 3. Effect of the volume of extraction solvent (CCl4 ) on the recovery. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL.
Fig. 5. Effect of the pH on the extraction of the compounds expressed as recovery. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL and extraction solvent (CCl4 ) volume, 60.0 L.
Fig. 4. Effect of the disperser solvent volume on the recovery and enrichment factor of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent, acetonitrile; extraction solvent, 40.0 L CCl4 .
3.1.3. Selection of the extraction solvent volume To examine the effect of the extraction solvent volume on the performance of the DLLME procedure, solutions containing different volumes of CCl4 were subjected to the procedure. The experimental conditions were fixed and included the use of 1.5 mL acetonitrile containing different volumes of CCl4 (10–100 L at 10L intervals). Fig. 3 shows the curve of recovery versus volume of extraction solvent (CCl4 ). With less than 30.0 L of carbon tetrachloride, no two-phase system was observed. By increasing the volume of CCl4 from 30.0 to 60.0 L, the extraction recovery was increased. This behavior can be explained because of the amount of analyte and the acetonitrile partitioning in CCl4 increased as the CCl4 volume increased, and led to the increase in efficiency. Above 60 L a decrease in efficiency was observed, probably due to the decrease in the ratio between the disperser and extraction sol-
Fig. 6. Effect of the speed of centrifuging on the recovery of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL; extraction solvent, 40.0 L CCl4 .
vent. The decreased ratio lowers the amount of droplets formation available for extraction, thereby lowering the extraction efficiency. Based on these observations, a volume of 60.0 L was used. 3.1.4. Selection of disperser solvent volume In order to study the effect of the disperser volume, the acetonitrile volume varied between 0.75 and 3.0 mL. At lower volumes of the disperser, tiny droplet formation may not be effective, the cloudy state may not be stable and may cause incomplete extraction. Additionally, lack of acetonitrile availability for partitioning in CCl4 phase may occur. Both phenomena will decrease the extraction efficiency. When the volume of the dispersive solvent is increased, the partitioned acetonitrile phase in CCl4 phase leads to the increase in the extraction efficiency. The results are
Table 2 Results of mass spectrometric conditions for the simultaneous analysis of pesticidea . Pesticide
Ionization mode
Precursor ion (m/z)
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
Carbofuran
ESI+
222
165 123
20 20
25 25
Clomazone
ESI+
240
125 100
30 30
25 15
Tebuconazole
ESI+
308
70 88
40 33
20 50
a
Dwell time 0.3 s.
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Fig. 7. Mass spectra with the chemical structure deduced for each of the selected fragments for (a) carbofuran (b) clomazone and (c) tebuconazole.
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shown in Fig. 4. By increasing the volume of acetonitrile, EF and R% are higher with 2.0 mL. Based on the results, 2.0 mL was chosen as an optimum volume for the disperser solvent. When volumes larger than 2.00 mL are used with 60 L of carbon tetrachloride, the formation of a sedimented phase was not observed. A possible explanation may be that, at high volumes, the solubility of pesticides in water increases and the volume of disperser solvent is so high that the extractor solvent is not separated from the disperser and the aqueous solution. No droplet was observed.
3.1.5. Effect of pH It is a common practice to acidify natural samples shortly after the collection in order to limit both abiotic and biotic degradation of organic contaminants [45], but the pH of aqueous solution which contains acidic or basic compounds should be controlled in the extraction process. The pH influences the ionization form of certain analytes; thereby, it affects their water solubility and extractability. In this study, the effect of the pH upon pesticides extractability with DLLME was investigated by varying the pH values from 2.0 to 6.0 (Fig. 5) with addition of H3 PO4 1:1 (v/v). Better extraction efficiency for all the compounds was observed at pH 2.0. It happens because, at lower pH values, the extraction is improved, probably due to the fact that, in this condition, analytes are largely neutral and the neutral form of the organic compound has a higher tendency to partition itself into the organic solvent if compared to the ionized form. So, by adding H3 PO4 to the solution, the analytes will be converted into molecular forms.
Table 3 Figures of merit in the DLLME and LC–ESI-MS/MS method. Pesticide
tR (min)
Linear range (g L−1 )
r
LOQs (g L−1 )
Carbofuran Clomazone Tebuconazole
0.87 1.19 1.69
1.0–1000 1.0–1000 1.0–1000
0.9990 0.9982 0.9992
0.02 0.02 0.02
Table 4 Recovery of the determination of pesticide residues of spiked water samples. Pesticide
Level of fortification (g L−1 )
Recovery (%)
RSD (%)
Carbofuran
0.02 0.1 0.2 2.0
120.0 74.8 77.4 62.7
7.2 2.8 9.1 1.9
Clomazone
0.02 0.1 0.2 2.0
75.1 73.4 76.2 79.8
6.1 5.7 5.7 3.2
0.02 0.1 Tebuconazole 0.2 2.0
100.6 82.3 78.0 63.7
5.8 4.7 7.8 3.1
3.2. MS/MS optimization parameters
3.1.6. Effect of the speed of centrifugation During centrifugation, the dispersed fine particles of extraction phase were sedimented in the bottom of the conical test tube. The speed of centrifugation was investigated on the recovery of the analytes. It can be observed in Fig. 6 that the value of 2000 rpm is sufficient to separate the phases and extract all the analytes. In speeds higher than 2000 rpm, the values of recovery decreased.
Results of mass spectrometric conditions for the simultaneous analysis of pesticides are shown in Table 2. All selected pesticides showed more efficient ionization in the positive mode. For each compound we selected the optimum collision energies with the aim of getting two characteristic MRM transitions with the best signal intensity. We chose the MRM transition with the best signal intensity for quantification and the second one for confirmation of the pesticide. Fig. 7 shows the characteristic product-ion mass spectra obtained in the positive mode, with the chemical structure deduced for each of the selected fragments. The mass spectra were obtained after fragmentation of the precursor ions, using the Daughter Scan acquisition mode.
3.1.7. Other parameters The effect of the extraction time on the DLLME of the analytes was also investigated. Extraction times of 0–10 min were tested. In DLLME extraction, time is defined as an interval time between the injection of the mixture of the disperser solvent and the extraction solvent, and before starting to centrifuge. This parameter did not show any significant influence on the recovery of analytes, indicating that extraction in DLLME occurs very fast. The salt addition to the sample may have several effects on the extraction efficiency. To investigate the influence of the ionic strength on the performance of DLLME, various experiments were performed by adding different NaCl amounts (0–5%) and keeping the other experimental conditions constant. The addition of salt increase the volume of the sedimented phase, probably because of the decrease in solubility of extraction solvent in the presence of salt; but the extraction recovery was almost constant. In this study, the volume of the sedimented phase was not important since the sedimented phase was evaporated and redissolved. For this reason, no salt was added to further experiments. We should point out that carbon tetrachloride as a sample solvent has no good chromatographic behavior in this study; therefore, after extraction, the organic phase is transferred to a test tube (with conical bottom) and dried under nitrogen stream. The residue is dissolved in methanol and injected into LC. In this procedure, a solvent exchange is performed. In order to obtain high EF, the volume of methanol used in the dissolution of analytes was 100.0 L.
Fig. 8. Chromatograms obtained from tap water samples: blank of water sample and water sample spiked with 0.2 g L−1 mixture of pesticides, in total ion chromatogram mode (TIC).
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Table 5 Comparison of DLLME with SPE method for the determination of these pesticides. Method
LOQ (g L−1 )
SPE DLLME
0.004 0.02
RSD (%) 2.7–20.7 1.9–9.1
Extraction time (min)
Sample volume (mL)
References
60 A few seconds
250 5
Caldas et al. [46] Represented method
3.3. Analytical performance Analytical characteristics were obtained under optimum conditions. The method which was used to quantify the substances of interest was external standardization using calibration curves, obtained by plotting peak area versus pesticide concentration. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method and linearity was observed within the range of 1.0–1000 g L−1 . A good linear correlation among the compound concentration and peak areas was found with coefficient correlations (r) values in the range of 0.9982–0.9992. The quantification limits (LOQs) were calculated based on a signal-to-noise ratio (S/N) of individual peaks, assuming a ratio of 10:1 to LOQ. After the 50-fold DLLME preconcentration step, the effective LOQ for the samples was 0.02 g L−1 . The preconcentration value of 50-fold was obtained by dividing the initial volume of 5 mL by the final volume of 100 L. The retention times, linear range for the analytes, linear correlation coefficient and the limits of quantification for DLLME and LC–ESI-MS/MS are shown in Table 3. To assess the performance of the established method, the extraction recoveries were performed by spiking water samples with a mixture solution of the pesticides. For each concentration level, three replicate experiments with the whole analysis process were made. The results are presented in Table 4. The recoveries of the method for the analytes are between 62.7% and 120% at the concentration levels from 0.02 to 2.0 g L−1 , and the RSDs ranged from 1.9% to 9.1%, showing that DLLME procedure can be applied prior to the chromatographic analysis, allowing pesticide quantification at 0.02 g L−1 level, as demanded by the strict legislation [14]. The method was also successfully used for the determination of three pesticides in tap water samples, and the chromatogram of water blank and spiked water samples can be observed in Fig. 8. 3.4. Comparison of DLLME with SPE A comparison was made between the currently proposed method and SPE, which is one of the most used techniques for pesticide extraction in aqueous samples. Table 5 illustrates some differences between the methods, and it can be concluded that DLLME has short extraction time, lower solvent and sample volume consumption. Although the SPE has limits of quantification lower than DLLME, the achieved limits were below the limits established by the strict legislation, such as the European Union’s [14]. 4. Conclusions A dispersive liquid–liquid microextraction procedure was presented for the extraction and concentration of pesticides from aqueous samples. DLLME provides high recovery, wide linearity and good repeatability within a very short time. In comparison with other conventional extraction methods, this method has advantages such as rapidity, simplicity, ease of operation, and lower consumption of organic solvent. The DLLME combined with LC–ESIMS/MS was successfully applied to the analysis of carbofuran, clomazone and tebuconazole in waters, indicating that the pro-
posed method is suitable for the determination of different classes of pesticides in real samples. Acknowledgments This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), and Fundac¸ão de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). References [1] D.A. Lambropoulou, T.A. Albanis, J. Biochem. Biophys. Methods 70 (2007) 195–228. [2] M. Kuster, M.L. Alda, D. Barceló, Mass Spectrom. Ver. 25 (2006) 900–916. [3] F. Hernández, J.V. Sancho, O. Pozo, A. Lara, E. Pitarch, J. Chromatogr. A 939 (2001) 1–11. [4] M.A. Bacigalupo, G. Meroni, J. Agric. Food Chem. 55 (2007) 3823–3828. [5] J.B. Baugros, B. Giroud, G. Dessalces, M.F. Grenier-Loustalot, C. Cren-Olivé, Anal. Chim. Acta 607 (2008) 191–203. [6] R.B. Schäfer, A. Paschke, B. Vrana, R. Mueller, M. Liess, Water Res. 42 (2008) 2707–2717. [7] R. Zanella, E.G. Primel, S.L.O. Machado, F.F. Goncalves, E. Marchezan, Chromatographia 55 (2002) 573–577. [8] E. Marchesan, R. Zanella, L.A. Avila, E.R. Camargo, S.L.O. Machado, V.R.M. Macedo, Sci. Agric. 64 (2007) 131–137. [9] E.C. Bortoluzzi, D.S. Rheinheimer, C.S. Gonc¸alves, J.B.R. Pellegrini, A.M. Maroneze, M.H.S. Kurz, N.M. Bacar, R. Zanella, Quim. Nova 30 (2007) 1872–1876. [10] L. Carbo, V. Souza, E.F.G.C. Dores, M.L. Ribeiro, J. Braz. Chem. Soc. 19 (2008) 1111–1117. [11] M.N. Marques, M.B. Cotrim, M.A.F. Pires, O.B. Filho, Quim. Nova 30 (2007) 1171–1178. [12] D.D. Grützmacher, A.D. Grützmacher, D. Agostinetto, A.E. Loeck, R. Roman, S.C. Peixoto, R. Zanella, R. Bras. Eng. Agríc. Ambiental 12 (2008) 632–637. [13] A.M. Rodrigues, V. Ferreira, V.V. Cardoso, E. Ferreira, M.J. Benoliel, J. Chromatogr. A 1150 (2007) 267–278. [14] EU Council, Directive on the Quality of Water Intended for Human Consumption, 98/83/EC, 1998. [15] D. Nagaraju, S.D. Huang, J. Chromatogr. A 1161 (2007) 89–97. [16] Y. Pico, M. Fernández, M.J. Ruiz, G. Font, J. Biochem. Biophys. Methods 70 (2007) 117–131. [17] Y. He, H.K. Lee, Anal. Chem. 69 (1997) 4634–4640. [18] O.S. Fatoki, R.O. Awofolu, J. Chromatogr. A 983 (2003) 225–236. [19] Y.R. Tahboub, M.F. Zaater, Z.A. Al-Talla, J. Chromatogr. A 1098 (2005) 150–155. [20] M. Rezaee, Y. Assadi, M.R.M. Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, J. Chromatogr. A 1116 (2006) 1–9. [21] J.L.R. Raposo Júnior, N. Ré-Poppi, Talanta 72 (2007) 1833–1841. [22] X. Xu, H. Yang, L. Wang, B. Han, X. Wang, F.S. Lee, Anal. Chim. Acta 591 (2007) 87–96. [23] M. García-López, I. Rodríguez, R. Cela, J. Chromatogr. A 1166 (2007) 9–15. [24] E. Psillakis, N. Kalogerakis, Trends Anal. Chem. 22 (2003) 565–574. [25] S. Igarashi, N. Ide, Y. Takagai, Anal. Chim. Acta 424 (2000) 263–269. [26] A.S. Yazdi, N. Razavi, S.R. Yazdinejad, Talanta 75 (2007) 1293–1299. [27] E. Zhao, W. Zhao, L. Han, S. Jiang, Z. Zhou, J. Chromatogr. A 1175 (2007) 137–140. [28] J. Xiong, B. Hu, J. Chromatogr. A 1193 (2008) 7–18. [29] S. Berijani, Y. Assadi, M. Anbia, M.R.M. Hosseini, E. Aghaee, J. Chromatogr. A 1123 (2006) 1–9. [30] M.A. Farajzadeh, M. Bahram, J.Å. Jönsson, Anal. Chim. Acta 591 (2007) 69–79. [31] P. Liang, L. Guo, Y. Liu, S. Liu, T. Zhang, Microchem. J. 80 (2005) 19–23. [32] G.H. Wei, Y.Y. Li, X. Wang, J. Sep. Sci. 30 (2007) 3089–3324. [33] Y. Li, G. Wei, J. Hu, X. Liu, X. Zhao, X. Wang, Anal. Chim. Acta 615 (2008) 96–103. [34] A.P. Birjandi, A. Bidari, F. Rezaei, M.R.M. Hosseinia, Y. Assadi, J. Chromatogr. A 1193 (2008) 19–25. [35] X. Zang, W. Wu, M. Zhang, G. Xi, Chin. J. Anal. Chem. 37 (2009) 161–168. [36] C.B. Ojeda, F.S. Rojas, Chromatographia 69 (2009) 1–13. [37] X. Zang, J. Wang, O. Wang, M. Wang, J. Ma, G. Xi, Z. Wang, Anal. Bioanal. Chem. 392 (2008) 749–754. [38] R.R. Kozani, Y. Assadi, F. Shemirani, M.R.M. Hosseini, M.R. Jamali, Chromatographia 66 (2007) 81–86.
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Validation of method for determination of different classes of pesticides in aqueous samples by dispersive liquid–liquid microextraction with liquid chromatography–tandem mass spectrometric detection Sergiane Souza Caldas, Fabiane Pinho Costa, Ednei Gilberto Primel ∗ Programa de Pós-Graduac¸ão em Química Tecnológica e Ambiental, Escola de Química e Alimentos, Universidade Federal do Rio Grande, Av Itália, km 8 s/n, Rio Grande, Rio Grande do Sul State, 96201-900, Brazil
a r t i c l e
i n f o
Article history: Received 27 November 2009 Received in revised form 2 March 2010 Accepted 6 March 2010 Available online 12 March 2010 Keywords: Dispersive liquid–liquid microextraction Pesticides Liquid chromatography–tandem mass spectrometry Sample preparation Aqueous samples
a b s t r a c t In this study, a simple, rapid and efficient method has been developed for the extraction and preconcentration of different classes of pesticides, carbofuran (insecticide), clomazone (herbicide) and tebuconazole (fungicide) in aqueous samples by dispersive liquid–liquid microextraction (DLLME) coupled with liquid chromatography–tandem mass spectrometric detection. Some experimental parameters that influence the extraction efficiency, such as the type and volume of the disperser solvents and extraction solvents, extraction time, speed of centrifugation, pH and addition of salt were examined and optimized. Under the optimum conditions, the recoveries of pesticides in water at spiking levels between 0.02 and 2.0 g L−1 ranged from 62.7% to 120.0%. The relative standard deviations varied between 1.9% and 9.1% (n = 3). The limits of quantification of the method considering a 50-fold preconcentration step were 0.02 g L−1 . The linearity of the method ranged from 1.0 to 1000 g L−1 for all compounds, with correlation coefficients varying from 0.9982 to 0.9992. Results show that the method we propose can meet the requirements for the determination of pesticides in water samples. The comparison of this method with solid-phase extraction indicates that DLLME is a simple, fast, and low-cost method for the determination of pesticides in natural waters. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Pesticides, including substances with high toxic effects and persistence in the environment, have been widely used in agriculture throughout the world. The pollution of environmental compartments involves a serious risk to the environment and to human health as well, due to either direct exposure or residues in food and drinking water [1,2]. Compounds belonging to different classes such as carbofuran (carbamate), clomazone (isoxazolidinone), and tebuconazole (triazole) have been widely used on a variety of crops in current agricultural practices worldwide; they have been found in natural waters in many regions of the world [3–6], and in Brazil [7–12]; therefore, it is important to have fast and simple methods to determine these compounds. Owing to the toxicity of pesticides, the US Environmental Protection Agency (EPA) and the European Union (EU) have included them in their list of priority pollutants [13]. The European Union (EU) establishes rigid limits for pesticides in water
∗ Corresponding author. Tel.: +55 53 32336956; fax: +55 53 32336961. E-mail addresses: [email protected], [email protected] (E.G. Primel). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.03.004
destined to human consumption, establishing 0.1 g L−1 for individual pesticides and 0.5 g L−1 for the sum of all pesticides [14]. Since the concentrations of these compounds are normally low (g L−1 or less), and due to the low detection levels required by regulatory bodies and the nature of the matrices in which the target compounds are present, efficient sample preparation and trace-level detection and identification are important aspects of analytical methods. New analytical methods that use less quantity of solvents and that are easy and rapid to execute are required for monitoring the widespread distribution of trace levels of pesticides [15,16]. Liquid–liquid extraction (LLE) is one of the oldest preconcentration and matrix isolation techniques in analytical chemistry [17], but it is still used in routine sample preparation for the determination of pesticides [18,19], not only because of its simplicity, robustness, minimal operator training, and efficiency, but also because of its wide acceptance in many standard methods [1]. However, conventional liquid–liquid extraction uses large amounts of solvent, which are often hazardous, and time-consuming to perform. Therefore, to overcome these disadvantages, techniques such as solid-phase extraction (SPE) have been developed. Solid-phase extraction is already a well-established and routine technique. SPE
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uses much less solvent than LLE, but can be relatively expensive [20]. Microextraction techniques, such as solid-phase microextraction (SPME), have been widely applied for the preconcentration and quantification of pesticides in water [21,22] and some of the potential advantages of microextraction techniques are miniaturization, reduction of organic solvent consumption and improvement in the selectivity of the extraction [23]. Solvent phase microextraction (single drop microextraction, liquid-phase microextraction, liquid–liquid microextraction, etc.), based on a traditional LLE technique, is a less known method that utilizes only a few microliters of organic solvent as the extracting phase [17,24]. Assadi and his co-workers have recently developed a new microextraction technique named dispersive liquid–liquid microextraction (DLLME) as a high-performance, rapid and inexpensive microextraction [20]. The basic principle of this method is the dispersion of extraction solvent (immiscible in water) assisted with disperser solvent (miscible in both water and extraction solvents) within an aqueous solution which generates a very high contact area between the aqueous phase and the extraction solvent. From a commercial, economic and environmental point of view, the advantages of the DLLME method over conventional solvent extraction methods are simplicity of operation, rapidity, low cost, easier manipulation, less amount of organic extraction solvents, high recovery and enrichment factor, and easier linkage to analytical methods [25], such as GC [15,26–28] and LC [29–32]. Its application has been extended to separation, preconcentration and determination of organic and inorganic compounds in liquid samples [15,23,33,34], but most studies of pesticides evaluated the extraction of compounds that belong to the same chemical group, with the same behavior [35,36]. An efficient technique for sample preparation should have the ability to extract compounds from different chemical classes in a single step. Therefore, the aim of this study is to examine the DLLME and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) suitability for the determination of carbofuran, clomazone and tebuconazole simultaneously in water samples. In addition, some important parameters, such as pH, kind and volume of extraction solvent and disperser solvent, extraction time, speed of centrifugation, and salt effect are investigated and optimized.
Other chemicals such as carbon tetrachloride, tetrachloroethylene, monochlorobenzene, 1,2-dichlorobenzene (spectroscopy grade) were purchased from Vetec (Rio de Janeiro, Brazil) and dichloromethane, chloroform (spectroscopy grade) and sodium chloride from Merck (Darmstadt, Germany). The LC-grade methanol, acetone and acetonitrile were bought from Mallinckrodt (Phillisburg, NJ, USA). Fresh tap water samples for the optimization of the method were collected in our laboratory. No filtration or any further treatment was conducted in tap water samples. For each batch of water samples, one blank water sample was analyzed.
2.2. Instruments Liquid chromatography with mass spectrometric detection was performed in a Waters Alliance 2695 Separations Module (Waters, Milford, USA) fitted with an autosampler, a membrane degasser and a quaternary pump. Mass spectrometry was performed on a Micromass Quattro Micro API (Waters, Milford, USA) with an ESI interface. The LC column was an XTerra 3.5 m particle size (50 × 3 mm i.d.) (Waters, Milford, MA, USA). Analytical instrument control, data acquisition, and treatment were performed by software Masslynx version 4.1 2005 (Micromass, Waters, Milford, MA, USA). A sample volume of 20 L was injected with an autosampler. The mobile phase was acetonitrile:water (52:48, v/v), acidified with 0.1% formic acid at a constant flow of 0.4 mL min−1 . Ionization of the compounds was made using electrospray interface (ESI) in the positive mode. The run time was 3 min. Typical interface conditions were optimized for maximum intensity of the precursor ions as follows: capillary voltage, 3.5 kV, nebulizer and desolvation (drying gas) flows were set at 350 and 150 L h−1 , respectively; source block and desolvation temperatures were 120 and 350 ◦ C, respectively. Nitrogen was used as nebulizing, desolvation and cone gas, and argon was used as collision gas. Optimization of the MS/MS conditions, i.e., choice of the ionization mode, identification of the parent and product ions, and selection of the cone and collision voltages, more favorable for the analysis of the target analytes, was performed with injection of their individual standard solutions. Both modes of ionization, negative and positive, were tested.
2. Experimental 2.1. Reagent and materials
2.3. Dispersive liquid–liquid microextraction procedure
Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7ylmethylcarbamate), clomazone (2-(2-chlorobenzyl)-4,4-dimethyl -1,2-oxazolidin-3-one) and tebuconazole ((RS)-1-p-chlorophenyl4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol) analytical standards (purity > 99%) were supplied by Sigma–Aldrich (São Paulo, Brazil) and phosphoric acid (85%) analytical grade, by Merck (Darmstadt, Germany). Water was purified with a Direct-Q UV3® (resistivity 18.2 M cm, Millipore, USA) water purification system (Millipore, Bedford, MA, USA). Individual pesticide stock solutions containing 1000 mg L−1 of the target compounds were prepared in methanol and stored at −18 ◦ C. Intermediate working standards mixtures in methanol, containing 100 mg L−1 for each pesticide, were prepared and used to prepare the working standard solutions containing 1.0, 0.1, 0.05, 0.01, 0.005, 0.001 mg L−1 ; these were used for spiking samples and for preparing the analytical curves. Working standard solutions were prepared monthly and were stored at −18 ◦ C, while the dilutions used for the analytical curves were prepared daily.
A 5.0 mL working standard solution (0.2 g L−1 ) was placed in a 10.0 mL glass tube with conical bottom. Acetonitrile (2.0 mL) as the disperser solvent, containing 60 L carbon tetrachloride as the extraction solvent, was rapidly injected into the sample solution by using a 5.0 mL syringe. In this step, a cloudy solution (water/acetonitrile/carbon tetrachloride) was formed in the test tube and the pesticides in the water sample were extracted into fine carbon tetrachloride droplets. The resultant cloudy solution was centrifuged for 5 min at 2000 rpm (Centribio 80-2B, Curitiba, PR, Brazil). After centrifuging, the dispersed fine droplets of carbon tetrachloride sedimented in the bottom of test tube (about 50 L). The volume of the sedimented phase was determined by a 100 L microsyringe. The sedimented phase was completely transferred to another tube using a 100 L LC syringe and, after evaporation of the solvent under nitrogen stream, the residue was redissolved in 100 L LC-grade methanol and injected into the separation system. All experiments were performed in triplicate and results are shown in plots and tables.
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2.4. Calculation of enrichment factor and extraction recovery The enrichment factor (EF) was defined as the ratio between the analyte concentration in the settled phase (Cset ) and the initial concentration of the analyte (Co ) in the aqueous sample [20,29]. EF =
Cset Co
(1)
The extraction recovery (R%) was defined as the percentage of the total analyte which was extracted in the settled phase. R% =
Cset × Vset × 100% Co × Vaq
(2)
where R%, Vsed , and Vaq are the extraction recovery, the volume of the sediment phase, and the volume of the aqueous sample, respectively [37]. 3. Results and discussion 3.1. Optimized dispersive liquid–liquid microextraction conditions In this study, DLLME combined with LC–ESI-MS/MS was used for preconcentration and determination of selected pesticides in aqueous samples. There are various parameters that affect the DLLME performance and efficiency, such as the kind and the volume of the extraction and the disperser solvents, the ionic strength, and the extraction time. These parameters were investigated and, then, the optimal conditions were selected. 3.1.1. Selection of the extraction solvent The choice of an appropriate extraction solvent plays the main role in this method in order to achieve good recovery, enrichment factor, and selectivity of the target compounds. The extraction solvent should demonstrate (a) good chromatographic behavior (it should not have elution strength higher than the mobile phase used in the separation system), (b) extraction capability of the interest compounds, (c) low solubility in water, and (d) should form a cloudy solution in the presence of a disperser solvent when injected into an aqueous solution (form very tiny droplets) [30,38]. The density of the extraction solvent is an important characteristic and should be known. The first DLLME work [20] and most studies that determine pesticide in waters uses solvents with higher density of water [32,39,40], but there is some works with modification of DLLME that uses solvents with lower density of water [41–43]. Among the solvents whose density is higher than water (mainly chlorinated solvents), monochlorobenzene (C6 H5 Cl) dichloromethane (CH2 Cl2 ), 1,2-dichlorobenzene (C6 H4 Cl2 ), chloroform (CHCl3 ), tetrachloroethylene (C2 Cl4 ) and carbon tetrachloride (CCl4 ) were examined in order to find the most suitable solvent for DLLME. In this study, combinations of 40 L of C2 Cl4 , CCl4 , CHCl3 , CH2 Cl2 , C6 H4 Cl2 and C6 H5 Cl with 1.5 mL of acetonitrile as disperser solvent were tested. Having CH2 Cl2 , C6 H5 Cl and CHCl3 as extraction solvents, a two-phase system was not observed with the disperser solvent when they were injected into 5.0 mL analytes solution in water. With CCl4 , C6 H4 Cl2 and C2 Cl4 as extraction solvents, a twophase system was formed and its sedimented phase could easily be removed with a microsyringe. The results revealed that CCl4 has the highest extraction efficiency (12.8–42.3%) in comparison with C6 H4 Cl2 (5.1–19.0%) and C2 Cl4 (3.0–21.4%) (Fig. 1). Probably, it happens because of the high density and water solubility among the other solvents that CCl4 shows (Table 1). Although the main characteristic of the extraction solvent is that it must show low solubility in water, because of the polar characteristics of the analytes, certain solubility in water
Fig. 1. Effect of different extraction solvents on the recovery of three pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL; extraction solvent, 40 L. Table 1 Physical properties of extraction solvents. Solvent
Density (g mL−1 )
Water solubility (g L−1 )
C6 H5 Cl C6 H4 Cl2 CH2 Cl2 CHCl3 CCl4 C2 Cl4
1.11 1.31 1.32 1.48 1.59 1.62
0.5 0.15 13.8 8.0 0.8 0.17
seems to have favored the extraction of compounds. Thereby, CCl4 was selected as the extraction solvent. 3.1.2. Selection of disperser solvent The miscibility of the disperser solvent in the organic phase (extraction solvent) and in the aqueous phase (sample solution) is the main point for the selection of the disperser solvent [20]. The disperser solvent should be miscible in water; it should also dissolve the extraction solvent. Acetone, acetonitrile and methanol, which have got this ability, were selected for this purpose. A series of sample solutions was studied, using 1.5 mL of each disperser solvent containing 40.0 L of extraction solvent (carbon tetrachloride). With CCl4 as extraction solvent, a two-phase system was formed with all three disperser solvents; in comparison with other disperser solvents, the recoveries for all analytes were higher using acetonitrile. In Fig. 2, recovery was plotted as a function of the type of disperser solvent. It can be concluded that acetonitrile has an advantage over the other solvents, showing more ability to cover the desired analytical spectrum in study than other solvents used. In other works, as performed by Fu et al. [39] and Liu et al. [44], acetonitrile was also the disperser solvent used for extraction of pesticides in water samples. For this reason, carbon tetrachloride and acetonitrile were chosen as extraction and disperser solvents, respectively, in the following studies.
Fig. 2. Effect of the disperser solvent on the recovery of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent volume, 1.5 mL; extraction solvent, 40.0 L CCl4 .
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Fig. 3. Effect of the volume of extraction solvent (CCl4 ) on the recovery. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL.
Fig. 5. Effect of the pH on the extraction of the compounds expressed as recovery. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL and extraction solvent (CCl4 ) volume, 60.0 L.
Fig. 4. Effect of the disperser solvent volume on the recovery and enrichment factor of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent, acetonitrile; extraction solvent, 40.0 L CCl4 .
3.1.3. Selection of the extraction solvent volume To examine the effect of the extraction solvent volume on the performance of the DLLME procedure, solutions containing different volumes of CCl4 were subjected to the procedure. The experimental conditions were fixed and included the use of 1.5 mL acetonitrile containing different volumes of CCl4 (10–100 L at 10L intervals). Fig. 3 shows the curve of recovery versus volume of extraction solvent (CCl4 ). With less than 30.0 L of carbon tetrachloride, no two-phase system was observed. By increasing the volume of CCl4 from 30.0 to 60.0 L, the extraction recovery was increased. This behavior can be explained because of the amount of analyte and the acetonitrile partitioning in CCl4 increased as the CCl4 volume increased, and led to the increase in efficiency. Above 60 L a decrease in efficiency was observed, probably due to the decrease in the ratio between the disperser and extraction sol-
Fig. 6. Effect of the speed of centrifuging on the recovery of pesticides. Extraction conditions: sample volume, 5.0 mL; disperser solvent (acetonitrile) volume, 1.5 mL; extraction solvent, 40.0 L CCl4 .
vent. The decreased ratio lowers the amount of droplets formation available for extraction, thereby lowering the extraction efficiency. Based on these observations, a volume of 60.0 L was used. 3.1.4. Selection of disperser solvent volume In order to study the effect of the disperser volume, the acetonitrile volume varied between 0.75 and 3.0 mL. At lower volumes of the disperser, tiny droplet formation may not be effective, the cloudy state may not be stable and may cause incomplete extraction. Additionally, lack of acetonitrile availability for partitioning in CCl4 phase may occur. Both phenomena will decrease the extraction efficiency. When the volume of the dispersive solvent is increased, the partitioned acetonitrile phase in CCl4 phase leads to the increase in the extraction efficiency. The results are
Table 2 Results of mass spectrometric conditions for the simultaneous analysis of pesticidea . Pesticide
Ionization mode
Precursor ion (m/z)
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
Carbofuran
ESI+
222
165 123
20 20
25 25
Clomazone
ESI+
240
125 100
30 30
25 15
Tebuconazole
ESI+
308
70 88
40 33
20 50
a
Dwell time 0.3 s.
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Fig. 7. Mass spectra with the chemical structure deduced for each of the selected fragments for (a) carbofuran (b) clomazone and (c) tebuconazole.
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shown in Fig. 4. By increasing the volume of acetonitrile, EF and R% are higher with 2.0 mL. Based on the results, 2.0 mL was chosen as an optimum volume for the disperser solvent. When volumes larger than 2.00 mL are used with 60 L of carbon tetrachloride, the formation of a sedimented phase was not observed. A possible explanation may be that, at high volumes, the solubility of pesticides in water increases and the volume of disperser solvent is so high that the extractor solvent is not separated from the disperser and the aqueous solution. No droplet was observed.
3.1.5. Effect of pH It is a common practice to acidify natural samples shortly after the collection in order to limit both abiotic and biotic degradation of organic contaminants [45], but the pH of aqueous solution which contains acidic or basic compounds should be controlled in the extraction process. The pH influences the ionization form of certain analytes; thereby, it affects their water solubility and extractability. In this study, the effect of the pH upon pesticides extractability with DLLME was investigated by varying the pH values from 2.0 to 6.0 (Fig. 5) with addition of H3 PO4 1:1 (v/v). Better extraction efficiency for all the compounds was observed at pH 2.0. It happens because, at lower pH values, the extraction is improved, probably due to the fact that, in this condition, analytes are largely neutral and the neutral form of the organic compound has a higher tendency to partition itself into the organic solvent if compared to the ionized form. So, by adding H3 PO4 to the solution, the analytes will be converted into molecular forms.
Table 3 Figures of merit in the DLLME and LC–ESI-MS/MS method. Pesticide
tR (min)
Linear range (g L−1 )
r
LOQs (g L−1 )
Carbofuran Clomazone Tebuconazole
0.87 1.19 1.69
1.0–1000 1.0–1000 1.0–1000
0.9990 0.9982 0.9992
0.02 0.02 0.02
Table 4 Recovery of the determination of pesticide residues of spiked water samples. Pesticide
Level of fortification (g L−1 )
Recovery (%)
RSD (%)
Carbofuran
0.02 0.1 0.2 2.0
120.0 74.8 77.4 62.7
7.2 2.8 9.1 1.9
Clomazone
0.02 0.1 0.2 2.0
75.1 73.4 76.2 79.8
6.1 5.7 5.7 3.2
0.02 0.1 Tebuconazole 0.2 2.0
100.6 82.3 78.0 63.7
5.8 4.7 7.8 3.1
3.2. MS/MS optimization parameters
3.1.6. Effect of the speed of centrifugation During centrifugation, the dispersed fine particles of extraction phase were sedimented in the bottom of the conical test tube. The speed of centrifugation was investigated on the recovery of the analytes. It can be observed in Fig. 6 that the value of 2000 rpm is sufficient to separate the phases and extract all the analytes. In speeds higher than 2000 rpm, the values of recovery decreased.
Results of mass spectrometric conditions for the simultaneous analysis of pesticides are shown in Table 2. All selected pesticides showed more efficient ionization in the positive mode. For each compound we selected the optimum collision energies with the aim of getting two characteristic MRM transitions with the best signal intensity. We chose the MRM transition with the best signal intensity for quantification and the second one for confirmation of the pesticide. Fig. 7 shows the characteristic product-ion mass spectra obtained in the positive mode, with the chemical structure deduced for each of the selected fragments. The mass spectra were obtained after fragmentation of the precursor ions, using the Daughter Scan acquisition mode.
3.1.7. Other parameters The effect of the extraction time on the DLLME of the analytes was also investigated. Extraction times of 0–10 min were tested. In DLLME extraction, time is defined as an interval time between the injection of the mixture of the disperser solvent and the extraction solvent, and before starting to centrifuge. This parameter did not show any significant influence on the recovery of analytes, indicating that extraction in DLLME occurs very fast. The salt addition to the sample may have several effects on the extraction efficiency. To investigate the influence of the ionic strength on the performance of DLLME, various experiments were performed by adding different NaCl amounts (0–5%) and keeping the other experimental conditions constant. The addition of salt increase the volume of the sedimented phase, probably because of the decrease in solubility of extraction solvent in the presence of salt; but the extraction recovery was almost constant. In this study, the volume of the sedimented phase was not important since the sedimented phase was evaporated and redissolved. For this reason, no salt was added to further experiments. We should point out that carbon tetrachloride as a sample solvent has no good chromatographic behavior in this study; therefore, after extraction, the organic phase is transferred to a test tube (with conical bottom) and dried under nitrogen stream. The residue is dissolved in methanol and injected into LC. In this procedure, a solvent exchange is performed. In order to obtain high EF, the volume of methanol used in the dissolution of analytes was 100.0 L.
Fig. 8. Chromatograms obtained from tap water samples: blank of water sample and water sample spiked with 0.2 g L−1 mixture of pesticides, in total ion chromatogram mode (TIC).
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Table 5 Comparison of DLLME with SPE method for the determination of these pesticides. Method
LOQ (g L−1 )
SPE DLLME
0.004 0.02
RSD (%) 2.7–20.7 1.9–9.1
Extraction time (min)
Sample volume (mL)
References
60 A few seconds
250 5
Caldas et al. [46] Represented method
3.3. Analytical performance Analytical characteristics were obtained under optimum conditions. The method which was used to quantify the substances of interest was external standardization using calibration curves, obtained by plotting peak area versus pesticide concentration. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method and linearity was observed within the range of 1.0–1000 g L−1 . A good linear correlation among the compound concentration and peak areas was found with coefficient correlations (r) values in the range of 0.9982–0.9992. The quantification limits (LOQs) were calculated based on a signal-to-noise ratio (S/N) of individual peaks, assuming a ratio of 10:1 to LOQ. After the 50-fold DLLME preconcentration step, the effective LOQ for the samples was 0.02 g L−1 . The preconcentration value of 50-fold was obtained by dividing the initial volume of 5 mL by the final volume of 100 L. The retention times, linear range for the analytes, linear correlation coefficient and the limits of quantification for DLLME and LC–ESI-MS/MS are shown in Table 3. To assess the performance of the established method, the extraction recoveries were performed by spiking water samples with a mixture solution of the pesticides. For each concentration level, three replicate experiments with the whole analysis process were made. The results are presented in Table 4. The recoveries of the method for the analytes are between 62.7% and 120% at the concentration levels from 0.02 to 2.0 g L−1 , and the RSDs ranged from 1.9% to 9.1%, showing that DLLME procedure can be applied prior to the chromatographic analysis, allowing pesticide quantification at 0.02 g L−1 level, as demanded by the strict legislation [14]. The method was also successfully used for the determination of three pesticides in tap water samples, and the chromatogram of water blank and spiked water samples can be observed in Fig. 8. 3.4. Comparison of DLLME with SPE A comparison was made between the currently proposed method and SPE, which is one of the most used techniques for pesticide extraction in aqueous samples. Table 5 illustrates some differences between the methods, and it can be concluded that DLLME has short extraction time, lower solvent and sample volume consumption. Although the SPE has limits of quantification lower than DLLME, the achieved limits were below the limits established by the strict legislation, such as the European Union’s [14]. 4. Conclusions A dispersive liquid–liquid microextraction procedure was presented for the extraction and concentration of pesticides from aqueous samples. DLLME provides high recovery, wide linearity and good repeatability within a very short time. In comparison with other conventional extraction methods, this method has advantages such as rapidity, simplicity, ease of operation, and lower consumption of organic solvent. The DLLME combined with LC–ESIMS/MS was successfully applied to the analysis of carbofuran, clomazone and tebuconazole in waters, indicating that the pro-
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