Journal of Analytical Science & Technology (2012) 3 (1), 113-120 Article www.jastmag.org DOI 10.5355/JAST.2012.

113

In-syringe dispersive liquid-liquid microextraction with liquid chromatographic determination of synthetic pyrethroids in surface water
Saeed S. Albaseer1, R. Nageswara Rao2*, Y.V. Swamy3, K. Mukkanti1
Centre for Chemical Sciences and Technology, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500085, India 2 HPLC group, Analytical Chemistry Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India 3 Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
1

*Corresponding author:
R. Nageswara Rao, Tel: +91-40-2719-3193, Fax +91-40-2716-0387, E-mail: rnrao55@yahoo.com

Abstract
An indigenously fabricated in laboratory glass syringe was used for in-syringe dispersive liquid-liquid microextraction (is-DLLME) and preconcentration of synthetic pyrethroids (SPs) from surface waters suitable for their determination by high performance liquid chromatography. In contrast to classical DLLME, is-DLLME allows the use of lighter-than-water organic solvents and the analysis of environmental samples without prior filtration. This feature is interesting due to the fact that SPs have strong affinity to adsorb onto solid particulates present in samples which can be then retained on filter paper. The effects of various parameters on the extraction efficiency were evaluated and optimized systemically using one-factor-at-a-time method (OFAT) and statistically using full factorial design. Three SPs, viz.; cypermethrin, resmethrin and permethrin, were analyzed as model SPs. The method showed good precision with RSD% in the range of 4.86.9. The method detection limits of the three SPs ranged from 0.14 to 0.16 ng mL-1. The proposed method was applied for the determination of SPs in lake and ground waters.

Key words: In-syringe, Dispersive liquid-liquid, Pyrethroids, Lake water, HPLC, Extraction

Introduction
Synthetic pyrethroids (SPs) are efficient insecticides
Received for review : 26/10/2011 Published on Web: 22/12/2011 Korea Basic Science Institute. All Rights Reserved.

and they are of increasing importance and use. Also, SPs have been replacing other classes of insecticides such as organophosphorous. SPs are of high selectivity in action with relative degradability in the environment [1]. However, their high toxicity to bees and aquatic organisms including fish with LC50 values <1.0 ppb [2]

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is of great concern. Although SPs are usually not sprayed onto water [3], they can enter lakes, ponds, rivers and streams with rainfall or runoff from agricultural fields. In addition, there are some indications of potential for pyrethroid-mediated endocrine effects [3]. Several methods have been reported for the determination of SPs in environmental matrices, some of which are used for routine analysis. In the recent years, however, miniaturized techniques for sample preparation have been paid great attention because they are eco-friendly, among other favourable features. Dispersive liquid-liquid microextraction (DLLME) is one of the latest introduced miniaturized sample preparation techniques. The classical DLLME, however, mandates that extraction solvent should be more dense than water. Most of the solvents used for conducting classical DLLME are either chlorocarbons or chlorohydro-carbons. These chlorinated solvents are potent central nervous system depressants or stimulants [4]. They also cause several other potential risks to humans and a number of chlorinated solvents have been shown to cause cancer in laboratory animals [5]. A few approaches have been reported for introducing devices which allow the use of lighter-than-water extraction solvents, either by using a narrow-necked glass tube [6], or by using a medical-vial-like vessel [7,8]. These devices, however, pose some practical inconvenience which can only be experienced during use. In the present work we have developed an in-syringe dispersive liquid-liquid microextraction (is-DLLME) method which allows the use of lighter-than-water extraction solvents. The use of the syringe is very convenient and practical. Is-DLLME has several advantages over the classical DLLME such as: i) toxicity of the extraction solvents is much lower than that of chlorinated solvents, ii) withdrawal of the organic layer is practically much convenient as it forms the upper layer in the sample mixture, and iii) isDLLME allows the extraction of SPs from surface water samples without prior filtration which is not possible in case of classical DLLME. In classical DLLME suspended solids present in the sample will precipitate to the tube bottom along with the extract making the extract injection very risky as it may cause column clogging. This is important as the revised European legislation on environmental quality

standards in the field of water policy considers only total concentrations in the whole water sample for assessment of organic contaminants [9]. In addition, SPs are known to adsorb strongly to solid particulates present in natural water samples [10,11], and, thus, determining the total SPs concentration mandates that such particulates should be included in the extraction process. For conducting is-DLLME, an indigenously fabricated in laboratory glass syringe was used. The effects of various experimental parameters on the extraction of cypermethrin (CYP), resmethrin (RES) and permethrin (PER) were optimized by means of onefactor-at-a-time method (OFAT) and statistically using full factorial design. The optimized parameters of the model were estimated by multiple linear regressions.

Materials and Methods

Pesticides and Reagents Permethrin (PER) and resmethrin (RES) (PESTANAL, analytical reagent grade, 98.2% and 98.5% purity, respectively) were obtained commercially (Sigma-Aldrich Co., USA), cypermethrin (CYP) (technical grade, 95% purity) was donated by Hyderabad chemicals (Hyderabad Chemicals Pvt. Ltd, Hyderabad, India). HPLC grade acetonitrile, methanol, and n-hexane and analytical reagent grade sodium chloride and cyclohexane were obtained from Merck (Merck Specialties Pvt. Ltd, Mumbai, India). HPLC grade toluene, ethyl acetate, and n-heptane were obtained from RFCL Ltd, India. All reagents were used without further purification. Instrumentation The chromatographic analysis was performed on an UPLC system (LC-20AT Prominence, Shimadzu, Japan) equipped with a binary solvent delivery system with inline degasser, an injection valve with a 20 L sample loop, a UV-diode array detector (model SPD-M 20A Prominence). The chromatographic separation was performed on a Phenomenex Luna HPLC column (250 4.6 mm, 5m particle size , Phenomenex, Torrance, CA, USA) filled with end capped RP-18 material. A ternary mixture mobile phase of acetonitrile: methanol: water having mixing ratio of 20:60:20 (v:v:v), respectively, was found optimal. The detection

Saeed S. Albaseer et al. 115

wavelength was set at 220 nm. Samples of 20 L volume were injected. The mobile phase flow rate was 1.00 mL min-1.

Results and Discussion

Optimization of is-DLLME using OFAT Factors affecting the extraction efficiency of isDLLME include type and volume of extractant and dispersant, extraction time, centrifugation speed and time, and salt addition. These factors were first optimized using one-factor-at-a-time method (OFAT) at several levels and the most effective two levels for each factor were then optimized statistically using full factorial design. OFAT is the most commonly used approach for method optimization which works by varying each factor at a time, while keeping all other factors at fixed levels. For method optimization, peak areas were used for assessing various factors. The extraction solvent suitable for is-DLLME should possess some essential properties which include: i) very low solubility in water, ii) high capability of extracting target analytes, and iii) less density than water. On the basis of these considerations, four organic solvents, viz.; n-hexane, cyclohexane, toluene and n-heptane were tested. OFAT experiments showed that the highest recoveries were obtained with n-hexane. The volume of hexane was, then, optimized. At volumes less than 80 L, bias increased substantially, and thus, n-hexane volume was assessed in the range of 80120 L. The results showed that n-hexane volumes of 100 and 120 L gave comparable recoveries although enrichment factors at 100 L n-hexane were higher. For is-DLLME, dispersant is an important parameter. This was proved when the method was conducted with and without adding a dispersant. For choosing the most suitable dispersant, three organic solvents, viz.; acetonitrile, methanol and acetone were tested. OFAT experiments showed that methanol was much inferior to acetonitrile and acetone in terms of dispersing efficiency. However, the results showed that acetonitrile and acetone had comparable efficiency and the differences lie within the error bars. Thus, it was decided to apply experimental design to evaluate the efficiency of acetonitrile and acetone as dispersant. The volumes of acetone and acetonitrile were tested at several levels and the most effective volumes were found to be 1.0 and 1.5 mL, respectively. In is-DLLME, extraction time was defined as the interval time after injecting the extraction mixture (the dispersant and the extractant) and before centrifugation.

Sample Preparation Stock solutions of cypermethrin (CYP), resmethrin (RES) and permethrin (PER) were prepared in HPLC grade acetonitrile (each 10 mg L-1) in amber reagent bottles and kept in the refrigerator at 4o C. Working reference solutions of concentrations ranged from 502000 ng mL-1 were prepared by dilution of the above stock solutions in HPLC grade acetonitrile and were used for construction of the calibration curve and preparation of spiked samples. Surface and ground waters were analysed for validating the optimized method. No filtration or any other treatment was conducted on water samples. Extraction Procedure For is-DLLME optimization, 5.0 mL double distilled water was placed in a 10 mL indigenously fabricated in laboratory glass syringe; the sample was spiked with the analytes and 0.25 mg NaCl (5 %, w/v) was added and dissolved. A mixture of 100.0 L n-hexane and 1.5 mL acetonitrile was rapidly added into the sample using a 2.0 mL glass syringe. The mixture was vigorously shaken for 5 min, during which, SPs were extracted into the fine droplets of n-hexane. The mixture was then centrifuged for 3 min at 5000 rpm (REMI centrifuge model R-24, REMI, Mumbai, India). The nhexane (containing the extracted analytes) formed the upper layer. 20 L of n-hexane layer was injected into the HPLC-PDA for analysis using a 25 L microsyringe (Hamilton, Switzerland), Fig. 1.

Fig. 1. Experimental set-up of in-syringe dispersive liquidliquid microextraction

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As the time required for injecting the extraction solution was almost constant and samples were immediately centrifuged after preparation at fixed time and speed, the only variable was shaking time. Shaking time was examined in the range of 05 minutes with other experimental conditions kept constant. In addition to mixing the mixture, shaking is also important due to the fact that SPs adsorb onto container walls [12]. Hence, tube shaking increases extraction efficiency by re-suspending the adsorbed analytes. The results showed that extraction time did influence the extraction efficiency of SPs. The shaking time, however, depends on the type of dispersant. Centrifugation time was also examined and 3-min centrifugation at speed of 5000 rpm was found optimum. Salting-out effect has been used universally in LLE to increase the extraction efficiency by decreasing the solubility of analytes in the aqueous phase and thus enhancing their partitioning into the organic phase. In this study, effect of salt addition on extraction efficiency was assessed and NaCl was chosen for this purpose. A series of experiments were performed with salt concentration in the range of 015 % (w/v). The OFAT experimental results showed that the recoveries of the analytes increased when NaCl was added at a concentration of 5 % (w/v). However, the analytes recoveries decreased when NaCl concentration was higher than 5%. The negative effect of higher

concentration of NaCl could be attributed to the excessive decrease in the analytes aqueous solubility leading to an increase in their adsorption onto tube wall. In addition to improving extraction efficiency, salt addition, also, prevented formation of emulsion which causes inconvenient withdrawal of floated phase.

Optimization of is-DLLME using DOE As discussed above, applying OFAT method showed that more than one level of each factor was found to be effective and the differences in efficiencies lie within the error bars. So, it was decided to apply design of experiment (DOE) method. DOE provides information about how factors interact in a way that OFAT method cannot. Based on OFAT results four factors at two levels each were studied using DOE. The factors and their levels were: type of dispersant (acetonitrile and acetone), volume of dispersant (1.0 and 1.5 mL), volume of extractant (80 and 100 L n-hexane), and shaking time (1 and 5 min). A full factorial design (24) was used to optimize the experimental conditions that affect the analyte response signals. The full factorial experimental matrix design with the experimental levels of the independent variables of is-DLLME method for CYP, RES and PER determination is given in Table 1. Multiple linear regression (MLR) was applied to estimate the parameters of the proposed model for each of the four variables.

Table 1. Experimental matrix design of the full factorial design of CYP, RES and PER determinations with is-DLLME. Factor name Factor code Level Low High Dispersant type Coded (A) Actual A1 A2 Acetone Acetonitrile Dispersant volume Coded (B) + Actual 1.0 mL 1.5 mL Extractant volume Coded (C) + Actual 80 L 100 L Shaking time Coded (D) Actual + 1.0 min 5.0 min

Fig. 2. Plot of normalized regression coefficient values, for scaled factors, obtained from MLR, for the three response variables (CYP, RES, PER) studied.

Saeed S. Albaseer et al. 117

A summary of these results is shown in Fig. 2, where the regression coefficient values for the studied factors are shown as bar graphs for all the responses. From this, it is possible to compare the coefficients between various responses. Fig. 2, also, shows the importance of the different terms in the model for each of the responses evaluated. Among the main factors effects, only shaking time had negligible effect which means that extraction took place very rapidly. However, most 2-way and 3-way interactions have stronger effects than

main factors. On the other hand, the obvious differences among the factors that influence the recovery of individual SPs come in agreement with previous reports which demonstrated that an individual SP may, in some analytical aspects, be considered as a disconnected, stand-alone entity. The ANOVA and the results of regression analysis of factorial design for the extraction of synthetic pyrethroids (SPs) from aqueous matrices using the optimized is-DLLME are given in Table 2.

Table 2. ANOVA and results of regression analysis of factorial design for pyrethroids extraction using is-DLLME Analyte CYP RES PER Factor Dispersant type (A) Dispersant volume (B) extractant volume (C) shaking time (D) A*B A*C A*D B*C B*D C*D 1: Coefficient value Coeff1. 0.0324011 0.016141 0.0502377 0.0084168 -0.0612961 0.0360974 -0.011456 0.0033693 0.0005973 0.0550756 Fvalue 0.7 0.35 1.08 0.18 -1.32 0.78 -0.25 0.07 0.01 1.18 Pvalue 0.613 0.787 0.476 0.886 0.413 0.58 0.846 0.954 0.992 0.447 Coeff. 0.02056 0.01824 -0.009 0.02174 -0.02394 0.02362 0.00203 -0.02186 0.0333 0.03576 Fvalue 1.2 1.06 -0.52 1.27 -1.4 1.38 0.12 -1.28 1.94 2.09 Pvalue 0.442 0.48 0.692 0.425 0.396 0.4 0.925 0.423 0.303 0.285 Coeff. 0.0028 0.01879 -0.00387 0.00831 -0.01861 0.04081 0.00567 0.0222 0.02781 0.04697 Fvalue 0.1 0.66 -0.14 0.29 -0.65 1.43 0.2 0.78 0.97 1.65 Pvalue 0.938 0.629 0.914 0.82 0.632 0.388 0.875 0.579 0.508 0.347

From the regression analysis of the results, the determination coefficient (R2) were calculated to be 85.9, 96.3 and 91.5 with (R2-adj) of 84.9, 96.0 and 90.9 for RES, PER and CYP, respectively. The plots of regression analysis are shown in Fig. 3. Contour plots for the response variables as a function of dispersant volume and shaking time and the contour plots for the response variables as a function of extractant volume and dispersant volume are shown in Fig. 4 (4a and 4b). By analyzing the plots for the SPs responses, keeping in mind that the ultimate goal was to maximize the extraction efficiency of is-DLLME, it was found that higher dispersant volume requires shorter shaking time and smaller extractant volume. This indicates the important role played by the dispersant in the extraction process by facilitating the release of analytes from the sample matrix into the extracting solvent. In addition, main effects plots (not shown) revealed that shaking

time factor had no effect by itself, although it plays a reasonable role when it interacts with other factors. In addition, from factor interactions plots (not shown) it was found that when acetonitrile was used as a dispersant, relatively longer shaking time ( 5 min) was required. This could be explained as a result of acetonitrile immiscibility in the extractant (n-hexane) resulting in relatively longer contact time for complete transfer of analytes into the extracting phase. A worth mentioning point is that, at optimum extraction conditions, the results showed that acetonitrile was relatively more efficient than acetone in terms of dispersing efficiency. As acetonitrile is immiscible with n-hexane, these results come in contrast with the unsubstantiated assumption that dispersant should be miscible in both aqueous sample and extractant, which was proposed when DLLME was first introduced [13]. This assumption was not yet evaluated as all solvents

118 Journal of Analytical Science & Technology (2012) 3 (1), 113-120

used as dispersants and extractants, so far, in classical DLLME are completely miscible with each other. Thus, we believe that, based on our results, the extraction mechanism in DLLME is not as simple as it was first proposed and further studies are required to explore the role of dispersing solvents.

Fig. 4. Contour plots for the pyrethroids recovery, as a function of (a) dispersant volume and shaking time and (b) dispersant volume and extraction volume.

Fig. 3. Plot of linear regression analysis of predicted values by full factorial analysis and experimentally observed values.

As can be inferred from the comments made above about the extraction conditions and the graphical plots of the results, design of experiments (DOE) offers a clearer picture on the optimum conditions for achieving higher extraction efficiency using the proposed sample preparation approach. Interestingly, slight differences in the optimum conditions for individual SPs were observed and, therefore, it was necessary to reach a compromise point. In conclusion, keeping in mind that 5 % NaCl (w/v) was applied to all runs during method optimization and the samples were centrifuged at 5000 rpm for 3 min, the optimum conditions predicted from the statistical analysis were: the dispersant type was acetonitrile at a volume of 1.5 mL and the extractant was n-hexane at a volume of 100 L with shaking time of 5 min. These predicted conditions were then verified experimentally and a good agreement between predicted an experimental results was achieved.

Saeed S. Albaseer et al. 119

Quantitative Analysis Analytical figures of merit were calculated according to ICH guidelines [14] under optimum experimental conditions. Linearity was assessed using samples spiked at five different concentration levels ranging from 1100 ng mL-1, except CYP, for which linearity was tested in the range of 110 ng mL-1 due to its low water solubility [15]. As shown in Table 3, good linearity was exhibited in the range of 110 ng mL-1, 1 64 ng mL-1 and 164 ng mL-1 with correlation coefficients (r2) of 0.9921, 0.9978 and 0.9969; and LODs of 0.16, 0.14 and 0.16 ng mL-1 for CYP, RES and PER, respectively. The recoveries of the method for the three insecticides were 98.7 %, 87.9 % and 89.7 %, with intra-day precision (expressed as RSD%) of 4.8, 6.9 and 6.2 and inter-day precision of 5.0, 7.8 and 7.3 (RSD%) for CYP, RES and PER, respectively, Table 4.
Table 3. Parameters of quantitative determination of SPs in water by is-DLLME. Analyte CYP RES PER Linearity (ng mL-1) 1.010 1.064 1.064 R2 0.9921 0.9978 0.9969 LOD (ng mL-1) 0.16 0.14 0.16

Table 4. Recoveries and enrichment factors of SPs in water by is-DLLME Analyte CYP RES PER Recovery (n=6) 98.7 86.9 89.7 Enrichment factor (n=6) 91 78 80.5 Intraprecision (RSD%) 4.8 6.9 6.2 Interprecision (RSD%) 5.0 7.8 7.3

hydrochloric acid to reduce its pH from 8.3 to 4.00 and then was kept in the refrigerator until analysis time. Analysis of unspiked samples showed that ground water was nearly free of contamination by the three pesticides or their levels were below the MDL of the proposed method. Cypermethrin (CYP), however, was detected in the lake water samples and was calculated to correspond to 1.3 ng mL-1. Although detected level of CYP was lower than the maximum limit legal established by the European Union (EU) for foodstuff and vegetables [16], it is greater than the LC50 values for fish (<1.0 ng mL-1) [2]. The detection of CYP may be attributed to its higher stability under natural conditions. Compared to other pyrethroids, CYP is relatively stable with a half-life of 816 days in direct sunlight as stated by National Pesticides Information Centre in its websites. In soil, Extension Toxicology Network [13] on its website, states that the half-life of CYP was found to be as long as 8 weeks, and in water as long as 100 days. Real water samples were then spiked at a concentration of 4.0 ng mL-1 and three replicates with the whole analytical process of the optimized method were performed. Recoveries of CYP, RES and PER were 97.4, 78.6 and 90.3 % for groundwater and 102.7, 85.1 and 93.2 % for lake water, respectively. Although lake water contains suspended particulates and organic matter which constitute additional surfaces for SPs adsorption, the efficiency of the method was not affected indicating that the method is capable of extracting solid particulates-bound SPs. Overlay chromatograms of unspiked and spiked lake water samples are shown in Fig. 5.

Analysis of Ground and Surface Waters To evaluate the efficiency and applicability of the proposed method to real samples, the extraction and determination of CYP, RES and PER in ground and lake waters were performed under optimized conditions. The ground water samples were collected from the lab and were analyzed immediately. Lake water sampling was done at an agricultural runoff-fed lake located about 30 km far from our lab. Lake water was collected in a glass container and kept in dark until it was brought to the lab where it was acidified with

Fig. 5. Chromatograms of a: unspiked lake water sample, and b: spiked lake water sample.

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Conclusions
A rapid and simple in-syringe DMLLE method followed by liquid chromatographic determination of three pyrethroids in water has been described. The proposed set up of is-DMLLE is efficient, inexpensive and easy to operate. The developed method allows the monitoring of pyrethroids in environmental water samples at concentration levels that can be toxic for aquatic organisms. The drawbacks of conventional DLLME utilizing denser-than-water extraction solvents are eliminated here.

Acknowledgement
The authors wish to thank Dr. J. S. Yadav, Director, Indian Institute of Chemical Technology, Hyderabad, for encouragement and permission to communicate the results for publication.

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