GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY

Optimization of dispersive liquidliquid microextraction of pyrethroid insecticides from aqueous samples for determination by reversed-phase high performance liquid chromatography
Saeed S. Albaseera, R. Nageswara Raob,*, Y.V. Swamyc, K. Mukkantia
Centre for Chemical Sciences and Technology, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500085, India b HPLC group, Analytical Chemistry Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India c Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
* a

Authors for correspondence: R. Nageswara Rao, email: rnrao55@yahoo.com Received 16 Mar 2010; Accepted 5 Apr 2010; Available Online 6 Apr 2011

Abstract Dispersive liquidliquid microextraction (DLLME) followed by high performance liquid chromatography with photo diode array detector (HPLC-PDA) has been evaluated for determination of three synthetic pyrethroids (SPs) in water. Adsorption of pyrethroid insecticides onto tube walls was one of its shortcomings. The effect of analyte adsorption was studied and a procedure to reduce pyrethroids loss onto tube walls was proposed. The effects of various variables on the extraction efficiency were evaluated and optimized. Under the optimum extraction conditions, the linearity was in the range of 0.2100 gL-1. Correlation coefficients varied from 0.9951 to 0.9992. Enrichment factors (EFs) ranged from 109 to 135. The recoveries of the investigated SPs range from 82.5% to 98.1% with RSD% values ranged from 5.6% to 10.8%. The limits of detection of the three pesticides ranged from 0.01 to 0.02 ng mL-1. Keywords: Dispersive liquid-liquid microextraction; Synthetic pyrethroids; Pesticides; Water analysis; Pyrethroids adsorption

1. Introduction Synthetic pyrethroid insecticides (SPs) are of increasing importance and use because of high selectivity in action, which could be attributed to the differences in uptake and distribution [1]. However, their high toxicity to bees and aquatic organisms including fish with LC50 values <1.0 ppb [2] is of great concern. SPs are usually not sprayed onto water [3,4] but they can enter lakes, ponds, rivers and streams from rainfall or runoff from agricultural fields. In addition, cypermethrin (CYP), resmethrin (RES) and permethrin (PER) (Figure 1) are regarded as endocrine disruptors [5]. Miniaturization in sample preparation is currently a major trend in analytical chemistry. Some typical examples of miniaturization techniques used for extraction of SPs from aqueous samples include solid phase microextraction (SPME) and liquid phase microextraction (LPME). However, SPME is expensive, its fiber is fragile, has limited lifetime and sample carry-over could be a problem [6]. On the other hand, LPME suffers from some
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disadvantages such as the possibility of formation of air bubble due to fast stirring, extraction is time-consuming and equilibrium could not be attained even after a long time extraction in most cases [7]. Rezaee et al. [8] introduced a microextraction technique which was referred to as dispersive liquidliquid microextraction (DLLME). DLLME is based on the formation of tiny droplets of the extractant in the sample solution using water-immiscible organic solvent (extractant) dissolved in a water-miscible organic dispersive solvent. Extraction of the analytes from aqueous sample into the dispersed organic droplets takes place. DLLME has been successfully applied to the determination of several contaminants in different matrices [9 12]. In this study, DLLME followed by high performance liquid chromatography (HPLC) with photo diode array detector (PDA) has been developed for the determination of three SPs in water samples. In addition, the effect of pyrethroids adsorption onto walls of tubes used to conduct DLLME was studied and was found to affect the extraction efficiency of DLLME.
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Figure 1. Chemical structures of the synthetic pyrethroids used.

The effect of various experimental parameters on the extraction of cypermethrin (CYP), resmethrin (RES) and permethrin was studied and discussed in detail. The optimized method was employed to investigate the levels of the target compounds in tap water samples. 2. Experimental 2.1. Chemicals and materials Permethrin (PER) and Resmethrin (RES) (PESTANAL, analytical reagent grade, 98.2% and 98.5% purity, respectively) were obtained from Sigma-Aldrich Co., USA. Cypermethrin (CYP) (technical grade, 95% purity) was donated by Hyderabad Chemicals Pvt. Ltd., Hyderabad, India. HPLC grade acetonitrile, methanol and acetone were obtained from MERCK (Merck Specialties Pvt. Ltd, Mumbai, India). Analytical reagent grade sodium chloride (NaCl) and ethanol were obtained from MERCK (Merck Specialties Pvt. Ltd., Mumbai, India). Analytical reagent grade carbon tetrachloride and tetrachloroethane were obtained from RFCL Pvt. Ltd., New Delhi, India. HPLC grade tetrachloromethane and chlorobenzene were obtained from MERCK (Merck Specialties Pvt. Ltd., Mumbai, India). All reagents were used without further purification. 2.2. Instrumentation The chromatographic analysis was performed on an UPLC system (LC-20AT
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Prominence, Shimadzu, Japan), equipped with a binary solvent delivery system with in-line degasser, an injection valve with a 20 L sample loop and a UV-diode array detector model SPDM 20A Prominence. The chromatographic separation was performed on a Phenomenex Luna C18, (250 x 4.6 mm I.D., 5 m particle size, Torrance-CA, USA). Mobile phase was acetonitrile - methanol - water, 20:60:20 (v/v). Ultra-pure water, purified by a Milli-Q water purification system, Millipore (Bedford, MA, USA) was used throughout the experiments unless stated, and was collected on daily basis and degassed with a vacuum pump and further filtered through 0.45 m membrane (Hydrophilic Nylon, Millipore). Samples of 20 l volume were injected at a mobile phase flow rate of 1.0 mL min-1. Diode array detector was set at 220 nm. A refrigerated centrifuge SIGMA 416K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany) was used for centrifugation. 15-mL polypropylene tubes with conical bottom as extraction vessel were purchased for the local market. 2.3. Sample preparation The stock solutions of the individual standard solutions and mixtures of the three SPs were prepared in pure HPLC grade acetonitrile in amber reagent bottles and kept in the refrigerator at +4o C. Working standard solutions of concentrations ranging from 502000 gL-1 were prepared by dilution of the above stock solutions
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Figure 2. Effect of nature of the extractant on extraction efficiency of SPs from water samples using DLLME.

in pure HPLC grade acetonitrile and used for constructing the standard calibration curve. Spiked samples in distilled water were prepared on daily basis and used for method optimization and validation. Tap water samples were collected from our lab and used without any treatment. 2.4. Extraction procedure For DLLME operations, 5.0 mL doubledistilled water was placed in a 15 mL screw cap polypropylene tube with conical bottom; the sample was spiked with the analytes and 0.25 mg NaCl (5%, w/v) was added and dissolved. A mixture of 65.0 L carbon tetrachloride (CCl4) and 1.0 mL acetonitrile was rapidly added into the sample using a 1.0 mL micropipette. A cloudy solution (water, acetonitrile and carbon tetrachloride) was immediately formed in the tube. Then, the mixture was gently shaken for 5 min by hand. In this step, SPs were extracted into the fine droplets of carbon tetrachloride. The mixture was then centrifuged for 3 min at 5000 rpm (refrigerated centrifuge, 27 C); the carbon tetrachloride phase was sedimented at the bottom of the centrifuge tube (~30 L). The upper aqueous solution was removed, using a 1.0 ml micropipette and 20 L of the sedimented phase was injected into the HPLC system for analysis using a 25 L microsyrange (Hamilton, Switzerland). 3. Results and Discussion 3.1. Optimization of DLLME Parameters affecting the extraction efficiency of DLLME include the type and volume of extractant and dispersant, extraction
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time and salt addition. In addition, a new parameter which is described here for the first time is the effect of tube pre-washing with the dispersant prior to performing DLLME as a way of reducing the effect of SPs adsorption onto tube walls. For preliminary experiments, extraction efficiency expressed as peak areas was used to assess various parameters. All experiments were performed in, at least, triplicates. 3.2. Selection of extraction solvent and effect of its volume In addition to properties such as very low solubility in water and high extraction capability of target analytes, the extraction solvent must be denser than water. In this experiment, carbon tetrachloride (CTC), tetrachloroethane (TCE), dichloromethane, and chlorobenzene (CB) were tested. Dichloromethane, however, was excluded as it did not form a separate organic phase even with injection of 100 L due to high water solubility. The other three solvents were tested and the best performance was obtained when carbon tetrachloride was used as the extraction solvent (Figure 2). The extractant volume was optimized over the range of 65-130 L and the experimental results showed that the enrichment factors decrease as the volume of the extractant increases as a result of dilution factor, although recovery calculations showed that various extractant volumes give comparable recoveries. However, for achieving higher enrichment factors and low detection limits (MDLs), the volume of 65 L was applied in the subsequent experiments.
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Figure 3. Effect of nature of the dispersant on extraction efficiency of SPs from water samples using DLLME.

3.3. Selection of dispersant and effect of its volume In the DLLME, the dispersant is an important parameter. In the preliminary experiments, several solvents were evaluated as dispersants. Results showed that acetonitrile (ACN) gives the highest extraction efficiency compared to acetone (ACE), methanol (MeOH) and ethanol (EtOH) (Figure 3). The volume of acetonitrile was then investigated over the range of 4001200 L and it was found that the enrichment factor increases as the amount of acetonitrile increases from 4001000 L beyond which, the extraction efficiency decreased. In this regard, it is worth mentioning that the effective comparison between different volumes of dispersant should take into account the change in the volume of the sedimented phase as a result of the change in the dispersant volumes which will be directly reflected in the enrichment factors, although the recoveries may be comparable. Hence, the variation of dispersant volume mandates that the extraction solvent volume should be adjusted to keep the volume of the sedimented phase almost constant. 3.3.1. Effect of extraction time In DLLME, extraction time is defined as the interval time after injecting the extraction mixture (dispersive solvent and extraction solvent) and before centrifugation. As the time required for injecting the extraction solution is almost constant and samples were immediately centrifuged after preparation at fixed time and rpm, the only variable was shaking time. Five
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shaking times, viz., 1, 3, 5, 10 and 15 min were examined with other experimental conditions kept constant. As shown in Figure 4, extraction time does influence the extraction efficiency of SPs and the extraction efficiency significantly increased from 1 to 5 min shaking time and no significant increase in extraction efficiency was observed beyond 5 min and hence a 5-min shaking time was applied during the subsequent method optimization and validation. The effect of shaking could be attributed to the fact that SPs are hydrophobic in nature and adsorb onto container surfaces or solids they come in contact with [1315]. Zhou et al. [16] reported that more than 60% of the SPs present in water samples were associated to walls of sample containers. Hence, tube shaking may decrease desorption rate by resuspending the adsorbed analytes. This comes in agreement with previous publications [17,18] which reported that shaking or agitation of water samples were effective tools to resuspend SPs associated to storage container walls. However, a 5-min shaking is still, by far, shorter than time required by other microextraction methods. In SPME, for instance, the equilibrium was not reached even with an 80min extraction time under fixed sub-optimal conditions [19]. 3.3.2. Effect of centrifugation time Several centrifugation times were tested and results showed that 3 min centrifugation time was optimum although only negligible difference between different centrifugation times was observed. Thus, the centrifugation step was

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Figure 4. Effect of shaking time on extraction efficiency of SPs from water samples using DLLME.

limited to 3 min in order to improve the productivity (sample throughput) of the method. 3.3.3. Effect of salt addition Salting-out effect has been used universally in LLE to increase the extraction efficiency due to decrease in the solubility of analytes in the aqueous phase and thus enhancing their partitioning into the organic phase. In this study, effect of salt addition was assessed on extraction efficiency and NaCl was chosen for this purpose. In this regard, it is worth mentioning that the effective evaluation of salt addition should take into account the change in the volume of the sedimented phase as a result of salting-out effect. Hence, volume of the extractant was simultaneously adjusted to keep the volume of the sedimented phase almost constant. A series of experiments was performed with salt concentration in the range of (010% (w/v). The experimental results showed that the relative peak areas of the analytes increased by increasing NaCl concentration up to 5% (w/v) and then decreased when the salt concentrations exceeded 5% (w/v) which could be attributed to increase analytes adsorption to the tube wall as a result of decreasing their aqueous solubility. Based on these results, 5% NaCl (w/v) was selected in subsequent experiments. The other benefit of salt addition is that it prevented formation of emulsion which causes inconvenient withdrawal of sedimented phase. 4. Analytes Adsorption Initial experiments of recovery studies showed that low recoveries of all analytes were obtained. In addition, a large bias was observed when the tubes were used more than once. This
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bias was more serious at low concentrations as the small difference between repeated injections makes a large difference in results. In addition, we observed that the degree of bias differs from analyte to another. Careful consideration of all steps of sample preparation showed that the most suspected source of bias and low recoveries may be the adsorption of analytes to the tubes walls. As mentioned in section 3.1.3, adsorption of SPs to container walls is a real challenge which may lead to reporting artificially low concentrations and rapid extraction methods may only minimize adsorption-derived errors but do not prevent it. In addition, studies showed that SPs adsorb to plastic containers more than glass containers [20,21]. In an attempt to minimize adsorption effect, Helmuth and others [22] pre-treated glass vials with a solution of high molecular weight, viz., poly(ethy1ene glycol) (M, 20 000; 4% w/v) which reduced the adsorption of SPs to vessel surfaces during sampling and storage. In the present work we revisited the approach but by simply prewashing the polypropylene tubes used for extraction with acetone or acetonitrile (dispersant) prior to addition of water sample into it. Figure 5 shows that tube pre-washing with acetonitrile or acetone improved extraction efficiency of all the analytes as a result of reducing the amount of analytes adsorbed onto tube walls. Compared to acetone, acetonitrile was more efficient. Prewashing effect could be attributed to the fact that some adsorptive sites on tube walls will be occupied by solvent molecules decreasing the interaction between the active sites and the analytes of interest, thus allowing a larger amount of the analyte to be present in the soluble form. Results also show that the degree of adsorption reduction was not
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Figure 5. Effect of tube prewashing on extraction efficiency of SPs from water samples using DLLME.

equal for all analytes. In addition, tube prewashing improved the repeatability of the method and the RSD% values decreased from about 24% to less than 10%. In conclusion, we recommend the following practice if DLLME is to be used for determination of SPs from aqueous samples: (i) prewashing tubes with a suitable organic solvent such as acetonitrile or acetone before transferring water sample into it; (ii) tubes should be used once only; (iii) sample shaking should be performed for at least five minutes. 5. Quantitative aspects Analytical performance of the optimized DLLME method was evaluated by HPLC-PDA. Analytical figures of merit were investigated under optimized experimental conditions and were calculated according to ICH guidelines [23]. Linearity was assessed using samples fortified at seven different concentration levels in the range from 0.2100 ng mL-1. Good linearity was exhibited and values of correlation coefficients (r2) were in the range of 0.9951 to 0.9992. The limits of detection (LODs), based on signal-to-noise ratio (S/N) of 3, ranged from 0.01 to 0.02 ng mL-1 (Table 1). Precision was

examined with samples spiked at three different concentrations within the above range i.e., 6, 24 and 48 ngmL1. For each concentration level, three replicate experiments with the whole analysis process were made and the results are given in Table 2. The recoveries (mean, n=9) of the method for the three insecticides in distilled water were high and recoveries of 98.1%, 82.5% and 89.6%, for CYP, RES and PER, respectively, were obtained. Relative standard deviations (RSDs) obtained under repeatability (intra-day precision) were below 10%. 6. Application to real samples To evaluate the efficiency and applicability of the proposed method to real samples, the extraction and determination of CYP, RES and PER in groundwater samples were performed. The water samples were collected from the main water supply of our lab and subsequently spiked with the standards of the three insecticides at concentration of 8.0 ng ml-1. Three replicate experiments with the whole analysis process of the optimized method were performed and the results are given in Table 3. The recoveries of the method (expressed as the mean percentage between the amounts found and

Table 1. Parameters of quantitative determination of SPs in water by DLLME.

Analyte CYP RES PER

Linearity range (ng mL-1) 0.5-100 0.2-100 0.5-100

R2 0.9992 0.9951 0.9964

LOD (ng mL-1) 0.01 0.02 0.01

LOQ (ng mL-1) 0.04 0.05 0.04

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Table 2. Recoveries and enrichment factors of SPs in water by DLLME.

Analyte CYP RES PER

Recovery (n=9) 98.1 82.5 89.6

Enrichment factor (n=9) 135.3 109.3 118.8

(RSD%) 8.5 5.6 10.8

Table 3. Analytical performance for DLLME for determination of SPs in tap water.

Analyte CYP RES PER

Spiked (ng mL1) 8 8 8

Found (ng mL1) stdev (n=3) 7.57 0.67 6.02 0.10 5.96 0.25

Recovery (n=3) 94.6 75.3 74.5

RSD (%) 8.3 1.7 2.87

the amounts added) for the three insecticides in tap water were 94.6, 75.2 and 74.5 for CYP, RES and PER, respectively. Figure 6 shows the typical chromatogram of the three analytes CYP, RES and PER in a tap water sample. To study the influence of the matrix on the proposed method, a calibration was realized by standard addition and the slopes were compared with those obtained by an external calibrate. A statistical comparison of standard deviation of the slopes was carried out according to Gonzlez et al. [24]. We calculated R as:

R=1. This must be checked for statistical significance:

R 1 u( R)

Where u(R) is the measurement uncertainty which is given by

u ( R)

u2 SAM b
2

2 b2 u b SAM

R b SAM

Where bSAM is the slope obtained by standard addition, b is the slope obtained by external calibration, The absence of matrix effect corresponds to bSAM=b, or, in terms of recovery,

According to the LGC/VAM protocol [25], t is compared with the two-tailed tabulated value, at the chosen % confidence level. If tttab, the consensus recovery is not significantly different from 1; If tttab,the recovery is significantly different from 1 and the analytical result must be corrected for R. For the three pyrethroids studied the value of t was

Figure 6. A typical HPLC chromatogram of the SPs extract from a spiked tap water sample obtained following the proposed DLLME procedure.
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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY inferior to 2. Therefore, it could be concluded that matrix effect does not affect the method efficiency. 7. Conclusions In the present work, the use of DLLME for SPs determination in water samples under the optimized conditions was demonstrated. The method gave satisfactory results and the procedure explained in this paper is easy to perform and improves the reliability of DLLME. Although SPs adsorption to sample tubes was a real problem, the procedure we have proposed is sufficient to provide reliable analytical results. Acknowledgment The authors wish to thank Dr. J. S. Yadav, Director, Indian Institute of Chemical Technology, Hyderabad, India, for his kind permission to communicate the results for publication. References 1. J. Stenersen, Chemical Pesticides: Mode of Action and Toxicology, CRC Press, Florida, USA (2004). 2. O.R. Daniel, L. Werner, White Paper for the Interagency Ecological Program, SFEI Contribution 415, San Francisco, CA (2005). www.up3project.org/documents/pyrethroids _white_paper_final.pdf 3. A. Mekebri, D.B. Crane, G.J. Blondina, D.R. Oros, J.L. Rocca, Bull. Environ. Contam. Toxicol. 80 (2008) 455. 4. G.D. Todd, D. Wohlers, M. Citra, Toxicological profile for pyrethrins and pyrethroids, ATSDR, U.S Department of Health and Human Services, Public Health Service, Atlanta, Georgia, USA (2003). 5. M. Zhao, F. Chen, C. Wang, Q. Zhang, J. Gan, W. Liu, Environ. Pollut. 158 (2010) 1968. 6. P. Li, Y.M. Qiu, H.X. Cai, Y. Kong, Y.Z. Tang, D.N. Wang, M.X. Xie, Chin. J. Chromatogr. 24 (2006) 14. 7. F. Ahmadi, Y. Assadi, M.R.M. Hosseini, M. Rezaee, J. Chromatogr. A 1101 (2006) 307. 8. M. Rezaee, Y. Assadi, M.R.M. Hosseini, E. Aghaee, F. Ahmadia, S. Berijani, J. Chromatogr. A 1116 (2006) 1. 9. L. Liu, J. Cheng, G. Matsadiq, H. Zhou, J. Li, Chromatographia 72 (2010) 1017. 10. H. Xu, D. Song, Y. Cui, S. Hu, Q.W. Yu, Y.Q. Feng, Chromatographia 70 (2009) 775. 11. H. Chen, H. Chen, J. Ying, J. Huang, L. Liao, Anal. Chim. Acta 632 (2009) 80. 12. R.S. Zhao, C.P. Diao, X. Wang, T. Jiang, J.P. Yuan, Anal. Bioanal. Chem. 391 (2008) 2915. 13. S.S. Albaseer, R.N. Rao, Y.V. Swamy, K. Mukkanti, J. Chromatogr. A 1217 (2010) 5537. 14. C.S. Brunetea, R.A. Prezb, E. Miguela, J.L. Tadeoa, J. Chromatogr. A 823 (1998) 17. 15. M.L. Hladik, K.L. Smalling, K.M. Kuivila, Bull. Environ. Contam. Toxicol. 80 (2008) 139. 16. L.J. Zhou, S. Rowland, R.F.C. Mantoura, Water Res. 29 (1995) 1023. 17. M.S. Sharom, K.R. Solomon, J. Agric. Food Chem. 29 (1981) 1122. 18. K. Day, N.K. Kaushik, Aquat. Toxicol. 10 (1987) 131. 19. P. Mayer, W.H.J. Vaes, J.L.M. Hermens, Anal. Chem. 72 (2000) 459. 20. C.E. Wheelock, J.L. Miller, M.J. Miller, B.M. Phillips, S.J. Geea, R.S. Tjeerdema, B.D. Aquat. Toxicol. 74 (2005) 47. 21. M.L. Hladik, J.L. Orlando, K.M. Kuivila, Scientific Investigations Report 20095012; U.S. Geological Survey, Reston, Virginia (2009). http://pubs.usgs.gov/sir/2009/5012/ 22. D.W. Helmuth, S.M. Ghiasuddin, D.M. Soderlund, J. Agric. Food Chem. 31 (1983) 1127. 23. Q2A, ICH, Q2 (R1), Validation of Analytical Procedures: Text and Methodology; International Conference on Harmonization, Geneva (2005). http://www.ich.org/LOB/media/MEDIA417. pdf. 24. A.G. Gonzlez, M.A. Herrador, Trends Anal. Chem. 26 (2007) 227. 25. V.J. Barwick, L.R. Ellison, VAM Project 3.2.1. Part d: Protocol for Uncertainty Evaluation from Validation Data, Report No: LGC/VAM/1998/088 (2000).

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