JAST
2012 March;3(1):113-120.
Published online 2011 December 22.
doi:http://dx.doi.org/10.5355/JAST.2012.113
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| Copyright ⓒ 2010 Journal of Analytical Science & Technology |
| 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 |
1CCST, Institute of Science and Technology, Jawaharlal Technological University Hyderabad Centre for Chemical Sciences and Technology
2*HPLC group, Analytical Chemistry Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
3Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India |
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Corresponding Author:
R. Nageswara Rao ,Tel: +91-40-2719-3193, Fax: +91-40-2719-3193, Email: rnrao55@yahoo.com |
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ABSTRACT |
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| 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 contaminants’ samples without prior filtration, which is of great importance due to the high affinity of pyrethroids to adsorb to solid particulates present in environmental samples. 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 (24). Three SPs (viz.; cypermethrin, resmethrin and permethrin) were analyzed. The method showed good accuracy with RSD% in the range of of 4.8–6.9%. The method detection limits of the three pesticides ranged from 0.14 to 0.16 ng mL-1. The proposed method was applied for the determination of synthetic pyrethroids in lake water |
| Keywords: In-syringe, Dispersive liquid-liquid, Pyrethroids, Lake water, Extraction, HPLC |
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INTRODUCTION |
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Synthetic pyrethroids (SPs) are efficient insecticides and they are of increasing importance and use as they have been replacing other classes of insecticides such as organophosphorous. SPs are of high selectivity in action and have relative degradability in the environment [1]. However, their high toxicity to bees and aquatic organisms including fish with LC50 values <1.0 ppb [2] 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 waters, some of which are used for routine analysis. In the recent years, however, miniaturized sample preparation samples have been paid great attention because they are eco-friendly. Dispersive liquid-liquid microextraction (DLLME) is one of the latest introduced miniaturized sample preparation techniques. The classical DLLME, however, mandates that the extraction solvent should be of higher density than water. Most solvents used for conducting classical DLLME are either chlorocarbons or chlorohydrocarbons. These chlorinated solvents are potent central nervous system depressants or stimulants. They also cause several other potential risks to humans and many have been shown to cause cancer in laboratory animals. 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 [4], or by using a medical-vial-like vessel [5,6], 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 and ii) the withdrawal of the organic layer is practically much convenient as it forms the upper layer in the sample mixture, and iii) is-DLLME allows the extraction of SPs from surface water samples without prior filtration which is not possible in case of classical DLLME as suspended solids present in the sample will precipitate to the tube bottom with the extract making the extract injection very risky as it may cause column clogging. This is of great importance 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 [7]. In addition, SPs are known to adsorb strongly to solid particulates present in natural water samples [8,9] 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 one-factor-at-a-time method (OFAT) and a full factorial (24) design. The parameters of the model were estimated by multiple linear regressions (MLR). The optimized method was employed for determination of the target compounds in ground and surface waters (lake water). |
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MATERIALS AND METHODS |
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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 obtained from Hyderabad chemicals (Hyderabad Chemicals Pvt. Ltd, Hyderabad, India). HPLC grade acetonitrile, methanol, and n-hexane were obtained from Merck (Merck Specialties Pvt. Ltd, Mumbai, India). Sodium chloride, cyclohexane, toluene, were all of analytical reagent grade (Merck Specialties Pvt. Ltd, Mumbai, India). HPLC grade toluene, ethyl acetate, and n-heptane were purchased from RFCL Ltd, India. All reagents were used without further purification.
Instrumentation
The chromatographic analysis was performed on a UPLC system (LC-20AT Prominence, Shimadzu, Japan) equipped with a binary solvent delivery system with inline degasser, an injection valve with a 20 FL sample loop, a UV-diode array detector (model SPD-M 20A Prominence), and a column oven (model CTO-10ASvp). The chromatographic separation was performed on a Phenomenex Luna HPLC column (250 × 4.6 mm, 5 μm particle size), (Phenomenex, Torrance, CA, USA) filled with end capped RP-18 material. A ternary mixture mobile phase of acetonitrile: methanol: water 20:60:20 (v:v:v) was found optimal. The detection wavelength was set at 220 nm. Samples of 20 FL volume were injected. The mobile phase flow rate was 1.00 mL min-1.
Sample Preparation
Stock solutions of cypermethrin (CYP), resmethrin (RES) and permethrin (PER) were prepared in HPLC gradient 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 50-2000 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 FL 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. In this step, 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 n-hexane phase was formed as an upper layer. 20 FL of the floated phase was injected into the HPLC-PDA for analysis using a 25 FL microsyringe (Hamilton, Switzerland), Fig 1. |
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RESULTS AND DISCUSSION |
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Optimization of is-DLLME using OFAT
In contrast to design of experiment (DOE), the most commonly used approach for method optimization is to vary each factor at a time, keeping all other factors at fixed levels. This approach is commonly known as one-factor-at-a-time method (OFAT). Parameters affecting the extraction efficiency of is-DLLME include the type and volume of extractant and dispersant, extraction time, centrifugation speed and time and salt addition. These factors were first optimized using OFAT method at several levels and the most effective two levels for each factor were then optimized statistically using a 24 full factorial design. For preliminary experiments, extraction efficiency expressed as peak areas was used to assess various parameters. The extraction solvent suitable for is-DLLME should possess some essential properties which include: i) very low solubility in water, ii) high extraction capability of target analytes and iii) lower 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 nhexane. The volume of hexane was, then, optimized; at volumes less than 80 FL, bias increases substantially, and thus n-hexane volume was assessed in the range of 80–120 FL. Results showed that 100 and 120 FL n-hexane give comparable recoveries although enrichment factors at 100 FL n-hexane were higher.
In the is-DLLME, the 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 the efficiency of methanol was much inferior to acetonitrile and acetone. However, the results showed that acetonitrile and acetone have 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 volume of the dispersant was tested at several levels and the most effective volumes were found to be 1.0 and 1.5 mL.
In is-DLLME, extraction time is defined as the interval time after injecting the extraction mixture (the dispersant and the extractant) 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 speed, the only variable is shaking time. Shaking time was examined in the range of 0–5 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 [10]. Hence, tube shaking increases extraction efficiency by re-suspending the adsorbed analytes. Our results showed that extraction time does 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 due to decrease of 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. A series of experiments were performed with salt concentration in the range of 0–15% (w/v). The OFAT experimental results showed that the relative peak areas of the analytes increase by increasing NaCl concentration up to 5% (w/v) beyond which the extraction efficiency decreases which could be explained as a result of excessive decrease in analytes aqueous solubility leading to an increase in analytes re-adsorption onto tube wall. In addition to improving extraction efficiency, salt addition prevents 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 was found to be effective and the difference in efficiencies lies 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. Factors and their levels are: type of dispersant (acetonitrile and acetone), volume of dispersant (1.0 and 1.5 mL), volume of extractant (and 100 FL n-hexane) and two levels of shaking time (1 and 5 min). A 24 full factorial design was investigated to optimize the analytical conditions that affect the analyte response signals. Table 1 shows the full factorial experimental matrix design, with the experimental levels of the independent variables (factors) of is-DLLME method for CYP, RES and PER determination. Multiple linear regression (MLR) was applied to estimate the parameters of the proposed model for each of the eight response variables. A summary of these results is shown in (Fig.2), where the regression coefficient values for studied factors are shown as bar graphs for all the responses considered. From this, it is possible to compare the coefficients between responses. (Fig.2), also, shows the importance of the different terms in the model for each of the responses evaluated. As can be seen most 2- way and 3-way interactions have stronger effects than main factors. Among the main factors effects, only shaking time has negligible effect which means that extraction takes place very rapidly. On the other hand, the obvious differences among the factors that influence the recovery of individual SPs come in agreement with previous reports which showed that indicate that an individual SP may, in some analytical aspects, be considered as a disconnected, stand-alone entity. Table 2 shows the ANOVA and results of regression analysis of factorial design for pyrethroids extraction using is-DLLME.
From the regression analysis of the results. The determination coefficient (R2) were calculated to be 86, 96.3 and 91.5 with (R2-adj) of 85, 96.0 and 90.9 for CYP, RES and PER, 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, and considering that, to maximize the extraction efficiency of is-DLLME, it is obvious that higher dispersant volume requires shorter shaking time and smaller extractant volume indicating the great role played by the dispersant in the extraction process by facilitating the release of analytes from the sample matrix into the extraction solvent. Main effects plots (not shown) tell us that shaking time factor has 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 can be deduced that, in general, when acetonitrile is used as a dispersant, relatively longer time (≤ 5 min) is required which can be explained by the fact that acetonitrile is immiscible in the extractant (n-hexane) and, hence, relatively longer contact time may become necessary for complete transfer of analytes into the extracting phase. A worth mentioning point is that at optimum extraction conditions results showed that acetonitrile gives relatively higher extraction efficiency than acetone. 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 the extractant, which was proposed when DLLME was first introduced [11]. This assumption was not later evaluated as all dispersants and extractants applied, so far, in classical DLLME are miscible with each other. Thus, we think, based on our results, that 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. 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 have been observed and, therefore, from the point of view of the whole process, it will be 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 as was obtained by OFAT method, the optimum conditions predicted from the statistical analysis are: the dispersant type is acetonitrile at 1.5 mL and the extractant is hexane at 100 FL with shaking time of 5 min. These predicted conditions were then verified experimentally and a good agreement between predicted an experimental results was observed.
Quantitative Analysis
Analytical figures of merit were calculated according to ICH guidelines [12] under optimum experimental conditions. Linearity was assessed using samples spiked at five different concentration levels ranging from 1–100 ng mL-1, except CYP, for which linearity was tested in the range of 1–10 ng mL-1 due to its low water solubility [13]. As shown in Table 3, good linearity was exhibited in the range of 1–10 ng mL-1, 1–64 ng mL-1 and 1–64 ng mL-1 with correlation coefficients (r2) of 0.9921, 0.9974 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 RSDs %values (intra-day precision) of 4.8, 6.9 and 6.2 and RSDs% values (inter-day precision) of 5.0, 7.8 and 7.3 for CYP, RES and PER, respectively, Table 4.
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 in an agricultural runoff-fed-lake located about 30 km far from our lab, and samples were collected in a glass container and kept in dark until it was brought to the lab where it was acidified with hydrochloric acid to reduce its pH from 8.3 to 4.00 and then was kept in the refrigerator until it was analyzed. 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 is lower than the maximum limit legal established by the European Union (EU) for foodstuff and vegetables [14], it is greater than the LC50 values for fish (<1.0 Fg L-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 8–16 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 concentration of 4.0 ng mL-1 and three replicates with the whole analysis 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 analytes adsorption, the efficiency of the method was not affected indicating that the method is capable of extracting adsorbed SPs and that matrix composition does not affect the efficiency of the method. (Fig.5). shows overlay chromatograms of a: unspiked lake water sample, and b: spiked lake water sample |
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CONCLUSIONS |
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| A rapid and simple in-syringe DMLLE method followed by liquid chromatography 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 denserthan- water extraction solvents are eliminated here. |
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Acknowledgement |
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| 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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FIGURES |
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Fig.1
Experimental setup of insyringe dispersive liquidliquid microextraction |
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Fig.2
Plot of normalized regression coefficient values, for scaled factors, obtained from MLR, for the three response variables (CYP, RES, PER) studied |
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Fig.3
Plot of linear regression analysis of predicted values by full factorial analysis and experimentally observed values |
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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 |
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Fig.5
Chromatograms of a: unspiked lake water sample, and b: spiked lake water sample |
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TABLES |
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Table.1
Experimental matrix design of the full factorial design of CYP, RES and PER determinations with is-DLLME. |
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Table.2
ANOVA and results of regression analysis of factorial design for pyrethroids extraction using is-DLLME |
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Table.3
Parameters of quantitative determination of SPs in water by is-DLLME. |
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Table.4
Recoveries and enrichment factors of SPs in water by is-DLLME |
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REFERENCE |
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| 1. |
Stenersen, J. Chemical Pesticides: Mode of Action and Toxicology; CRC Press: Florida, 2004; pp 116-121. |
| 2. |
Daniel, O. R.; Werner, L. Pyrethroid Insecticides: an analysis of use patterns, distributions, potential toxicity and fate in the Sacramento-San Joaquin delta and central valley; The Interagency Ecological Program, SFEI Contribution 415: San Francisco, 2005. |
| 3. |
Todd, G. D.; Wohlers, D.; Citra, M. Toxicological profile for pyrethrins and pyrethroids; ATSDR, U.S Department of Health and Human Services, Public Health Service: Atlanta, 2003. |
| 4. |
University of Oregon, Human resources. http://hr.uoregon. edu/policies-leaves/general-information/drug-alcohol-risks (accessed June 2011) |
| 5. |
David, P. D.; JOEL C. G. Occupational health the soldier and the industrial base, Office of The Surgeon General, U.S. Department of the Army Falls Church, Virginia, U.S. 1993, pp 469?170. |
| 6. |
Hashemi, P.; Beyranvand, S.; Mansur, R. S.; Ghiasvand, A. R. Development of a simple device for dispersive liquid-liquid microextraction with lighter than water organic solvents: Isolation and enrichment of glycyrrhizic acid from licorice. Anal. Chim. Acta. 2009, 655, 60?65. |
|
  |
| 7. |
Farajzadeh, M. A.; Seyedi, S. E.; Shalamzari, M. S.; Bamorowat, M. Dispersive liquid-liquid microextraction using extraction solvent lighter than water. J. Sep. Sci. 2009, 32, 3191?3200. |
|
|
| 8. |
Cruz-Vera, M.; Lucena, R.; Cardenas, S.; Valcarcel, M. One-step in-syringe ionic liquid-based dispersive liquid-liquid microextraction. J. Chromatogr. A 2009, 1216, 6459?6465. |
|
|
| 9. |
European Directive, 2008/105/EC of the European Parliament and of the Council on environmental quality standards in the field of water policy. Official J. Eur. Union, 2008, L 348, 84?97. |
|
|
| 10. |
Hladik, M. L.; Smalling, K. L.; Kuivila, K. M. A multi-residue method for the analysis of pesticides and pesticide degradates in water using HLB solid-phase extraction and gas chromatography-ion trap mass spectrometry. Bull. Environ. Contam. Toxicol. 2008, 80, 139?144. |
|
|
| 11. |
Zhou, L. J.; Rowland, S.; Mantoura, R. F. C. Partition of synthetic pyrethroid insecticides between dissolved and particulate phases. Water Res. 1995, 29, 1023?1031. |
|
|
| 12. |
Albaseer, S. S.; Rao R. N.; Swamy Y. V.; Mukkanti, K. An overview of sample preparation and extraction of synthetic pyrethroids from water, sediment and soil. J. Chromatogr. A 2010, 1217, 5537?5554. |
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| 13. |
Rezaee, M.; Assadi, Y.; Hosseini, M. R. M.; Aghaee, E.; Ahmadia, F.; Berijani, S. Determination of organic compounds in water using dispersive liquid-liquid microextraction. J. Chromatogr. A 2006, 1116, 1?9. |
|
|
| 14. |
Q2A, ICH, Q2 (R1). Validation of Analytical Procedures: Text and Methodology; International Conference on Harmonization: Geneva, 2005. |
| 15. |
Extension Toxicology Network, Pesticide Information Profiles. http://extoxnet.orst.edu/pips/cypermet.htm (Accessed on June 2011). |
| 16. |
The European Communities (Pesticide Residues) Regulations, (EU) No 520/2011. Official J. Eur. Union 2011, L140, 2?47. |
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