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Paula Grossi Igor R. B. Olivares Diego R. de Freitas Fernando M. Lancas

Laboratory of Chromatography, Institute of Chemistry at Sao Carlos, University of Sao Paulo, Sao Carlos, Brazil

Original Paper A novel HS-SBSE system coupled with gas chromatography and mass spectrometry for the analysis of organochlorine pesticides in water samples
A methodology to analyze organochlorine pesticides (OCPs) in water samples has been accomplished by using headspace stir bar sorptive extraction (HS-SBSE). The bars were in house coated with a thick film of PDMS in order to properly work in the headspace mode. Sampling was done by a novel HS-SBSE system whereas the analysis was performed by capillary GC coupled mass spectrometric detection (HSSBSE-GC-MS). The extraction optimization, using different experimental parameters has been established by a standard equilibrium time of 120 min at 858C. A mixture of ACN/toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. Reproducibility between 2.1 and 14.8% and linearity between 0.96 and 1.0 were obtained for pesticides spiked in a linear range between 5 and 17 ng/g in water samples during the bar evaluation.
Keywords: Headspace / Mass spectrometer / Organochlorine pesticides / Stir bar / Water analysis / Received: June 9, 2008; revised: August 7, 2008; accepted: August 8, 2008 DOI 10.1002/jssc.200800338

1 Introduction
The determination of pesticide residues in environmental samples, e. g., water and soil, as well as in agricultural products has been a major subject for many years because of their potential risk for human health, persistence, and tendency to bio-accumulate. In Brazil, as elsewhere, organochlorine pesticides (OCP) were used to control pests and thus improving crop yields during the 1970s. Included in the group of OCP pesticides were DDT, HCH, heptachlor, aldrin, dieldrin, and endrin. Although their use has been discontinued in Brazil since 1985, their persistence has left residual amounts in many areas. Analytical methods generally require extraction and enrichment steps before an analyst can perform the chromatographic separation and detection of trace organic

Correspondence: Professor Fernando M. Lancas, Laboratory of Chromatography, Institute of Chemistry at Sao Carlos, University of Sao Paulo, 13566-590 Sao Carlos, Brazil E-mail: flancas@iqsc.usp.br Fax: +55-16-3373-9983 Abbreviations: HS, headspace; LD, liquid desorption; OCP, organochlorine pesticides; SBSE, stir bar sorptive extraction; SPE,

compounds in aqueous matrices. During the extraction and enrichment steps, the trace amount solutes are isolated from the matrix and concentrated to enable their identification. In environmental, biomedical, and other types of analyses, the analyst uses a variety of extraction and enrichment techniques, including liquid liquid extraction (LLE) and SPE techniques. However, LLE is time consuming, generally labor intensive, and requires large quantities of expensive, toxic, and environmentally unfriendly organic solvents. The conventional offline SPE technique requires less solvent, but is relatively expensive [1, 2]. Modern approaches for sample preparation are more devoted to the solventless extraction methods, and miniaturization plays an important role in analytical chemistry. In the last few years, Pawliszyn and Arthur [3] developed the solid-phase microextraction (SPME), which is growing in popularity due to its ease of use, high sensitivity, and reproducibility. SPME has shown application in different areas like pharmaceutical analysis [4 8], food [9], pesticides [10 13], and fungicide [14], among others. More recently, stir bar sorptive extraction (SBSE) [15], a sample-preparation technique based on the same principles as SPME, has also been developed for the enrichment and sensitive determination of priority organic microwww.jss-journal.com

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pollutants in water samples [16 23] and other matrices [24 28]. In SBSE, the amount of PDMS typically coated, 24 126 lL, is substantially higher than that on an SPME fiber, for which the maximum volume is usually 0.5 lL (100 lm film thickness). This larger volume of PDMS results in higher recoveries and higher sample capacity. The SBSE technique was first introduced by Baltussen et al. [15] to extract organic analytes from aqueous samples. Sorptive extraction was almost contemporarily applied to headspace (HS) sampling by Tienpont et al. [29] and Bicchi et al. [30]. Sampling in the HS presents a significant advantage in terms of selectivity because only volatile and semivolatile organic compounds can be released into the HS. Since the phase is not in contact with the sample, background adsorption and matrix effects can be reduced, which also enhances the bar life expectancy. Using the same principle, HS-SPME has been used to determine volatile and semivolatile pesticide compounds in water samples and biological fluid samples showing good performance [31 39]. SBSE has been used in conjunction with thermal desorption (TD) then GC to enhance sensitivity. Although high performance is usually achieved, the TD technique is a one-shot process and when the sample has been desorbed there is no opportunity for method development and/or possible re-analysis. This great limitation can be overcome by liquid desorption (LD) which, in addition to being easy to use and cost effective, eliminates the need for expensive TD devices [40, 41]. The in-house SBSE development is important not only for cost reduction of the bars, but also to produce bars with different materials and thicknesses having specific characteristics which are more adequate for distinct analyses requirements. In this paper, the bars were in-house coated with a tick film of PDMS in order to properly work on HS mode. Sampling was done by holding the bar suspended in the HS, which is afterwards desorbed in a back-extraction solvent and injected into the GC-MS. Back-extraction using solvents instead of TD has the advantage of lowering the cost of the analyses, but the solvent used must be optimized to extract the compounds without affecting the bar. The extraction has been applied for the determination of 13 OCPs (a HCH; b HCH; c HCH; D HCH; heptachlor; aldrin; heptachlor epoxide; a endosulfan; DDE; dieldrin; endrin; DDD; and DDT) in water samples. In the present work, in order to evaluate the developed bars and considering the wide use of OCPs in the past, their toxicity, and the importance of their analyses, a methodology based on SBSE was developed and optimized. During method development the influence of different parameters in the extraction were studied, including temperature, extraction time, solvent polarity, and ultrasonic desorption time.

2 Experimental
2.1 Reagents and materials
ACN, toluene, ethyl acetate, methanol, and dichloromethane (all analytical grade) were purchased from Mallinckrodt (Phillipsbourg, NJ, USA). Sodium chloride (analytical reagent grade) was purchased from Merck (Darmstadt, Germany). OCPs of analytical standards (purity A98%) were purchased from Supelco (Bellefonte, PA, USA). Deionized water was obtained from Milli-Q purification system (Millipore, Milford, MA, USA). PDMS Sylgard 184 and cure agent were from Dow Corning Corporation (Midland, USA). Individual stock solutions (100 lg/mL) were prepared in toluene. A standard mixture containing 5 lg/mL of each pesticide in toluene, was prepared for use as spiking solutions. The working solutions were prepared by placing appropriate amounts of the standard mixture in an HS vial, the toluene was evaporated under purified nitrogen and afterwards redissolved in 15 mL of water.

2.2 Extraction
The bars were in-house coated with a thick film of PDMS in order to properly work on HS mode. The PDMS layer has a volume of 35 lL. The SBSE extractions were performed by holding the bar suspended in the HS, and by placing the spiked water into 20 mL HS vials capped with PTFE-coated septa. The samples (15 mL) were immersed in a temperaturecontrolled water bath from Quimis (So Paulo, Brazil) during the sampling process. The SBSE equilibrium was conducted at 600 rpm in a magnetic stirrer from VWR Scientific Products (USA) for an appropriate time period, during which analytes sorbed on the stationary phase coated over the bar. After extraction, the bar was desorbed in a back-extraction solvent into a 1.5 mL glass vial and the extraction was performed using different solvents. After all, an aliquot of 2 lL was injected into the GC-MS.

2.3 Instrument conditions

The GC-MS used was a Shimadzu QP5000 (Shimadzu, Japan). Separation was performed using a 30 m60.15 mm60.25 lm column NST-5 (5% phenyl95% methyl polysiloxane from NST, So Carlos, Brazil). The GC oven temperature was held for 5 min at an initial temperature of 1008C; then ramped at 158C/min to 2148C (4 min); ramped at 0.78C/min to 2298C; and finally ramped at 68C/min to 2648C. The carrier gas (helium)
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Figure 1. (a) Magnetic bar and the Teflon mold used for the coating procedure, (b) stir bar already coated. (c) Two parts of the mold and a ruler for dimensions comparison.

flow rate was operated in a constant flow mode at 1.1 mL/min. The interface temperature was 2808C. The mass spectrometer was operated in SIM mode and the ionization was done by electron ionization at 70 eV.

3 Results and discussion

3.1 Stir bars coating procedure
A Teflon mold was developed (Fig. 1) to cover a magnetic bar with PDMS, to be used during the extraction process. PDMS utilized consists of two materials, one being viscous phase and the other a cure agent. For the preparation of the bars different mixtures of the two components were tested to form the polymer in the desired consistency. The best proportion was 10:1 w/w. The viscous phase (0.3 g) was vigorously mixed with 0.03 g of the cure agent in a glass flask. Then the glass flask was placed under vacuum for 15 min to eliminate the bubbles formed during the homogenization process. The polymer and the magnetic bar were then poured in the mold. The mold was pressed with a steel support, using manual adjustment, to avoid leaks. Different temperatures and time were evaluated in order to obtain a consistent and homogeneous bar; the best results are described below. The mold with the bar was taken to a GC oven for 1 h at the temperature of 608C. After this the mold was cooled to room temperature and the coated bar was withdrawn from the mold and then placed in a Teflon holder. Finally, it was returned back to the oven using the follow-

ing temperature program: 408C (held for 30 min) and ramp of 108C/min to 2508C (held for 120 min). The bars were in-house coated as illustrated in Fig. 1. Figure 1a shows a photograph of the magnetic bar and the Teflon mold used for the coating procedure; Fig. 1b shows the bar already coated while Fig. 1c displays the two parts of the molding ensembly and a ruler for dimensions comparison.

3.2 Home-made SBSE parameters optimization

The number of research data has been increasing gradually in recent years due to instrumentation that allows a faster response. Thus, the application of statistics tools is of fundamental importance, particularly to explore and understand the data acquired. Experimental design is a technique used to define which data, in what quantity, and under what conditions should be collected for a given experiment, seeking essentially to meet two major objectives: the largest possible statistical accuracy in the response, and lower cost [42 47]. Several papers dealing with quantitative chromatography describe two main stages of experimentation: factor screening and response optimization. Two-level factorials and their fractions are frequently used for screening by different authors. In such studies the ranges of the variables are often well established and can be taken to vary between 1 and 1 [48, 49]. After preliminary, univariate studies evaluating the capabilities of the effects of temperature, time, desorption, and salinity were evaluated before the two-level facwww.jss-journal.com

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Figure 3. Pareto chart obtained for HS-SBSE optimization.

Figure 2. Response surface plot for (a) analytical signal versus sample temperature (8C) and extraction time (min), (b) analytical signal versus extraction time (min) and desorption time (min), and (c) analytical signal versus extraction time and ionic strength (NaCl%).

torial design to obtain the optimal conditions for HSSBSE. The experimental factors that were evaluated included: the extraction time (60 240 min), temperature (60 858C), NaCl added (0 10%), and desorption time (5 20 min). A fractional factorial design 24 1 was carried out to determine the influence of the selected factors and their interactions (Table 1). The response, based on the sum of peak areas, is one of the most useful parameters for the optimization. An analysis of variance (ANOVA) was performed to determine whether the experimental factors studied were significant (at a p-value of 0.05) on the performance of HS-SBSE system. From Fig. 2, it can be observed that the variables extraction time, temperature, desorption time, ionic strength, and the interactions between the variables, were highly significant. Figure 2 shows 3-D response surface graphs obtained by plotting different parameters. In Fig. 2a, the extraction time versus extraction temperature is presented. Figure 2b shows the response surface built for extraction time and desorption time, while Fig. 2c shows the response surface for extraction time versus ionic strength. The results showed that by increasing these factors, it will result in an increase in the analytical signal (Fig. 3). However, the factorial study also demonstrates that NaCl added did not significantly improve the extraction. By analyzing the response surfaces, the optimum conditions, inside the experimental domain that generates the highest extraction recovery were determined. These conditions correspond to extraction temperature of 858C, extraction time 120 min, and desorption time 15 min. In previous studies [22 24], specific experimental conditions have been shown to have substantial effect on the performance of the SBSE-LD when monitoring several classes of compounds in aqueous matrices. Based on this information the solvent effect was also evaluated in the proposed HS-SBSE system. LD solvents as MeOH, ACN,
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Table 1. Experimental design conditions studied Variables Extraction time (min) Temperature (8C) Ionic strength (% NaCl) Desorption time (min) Experiment 1 2 3 4 5 6 7 8 Extraction time + + + + Low level ( ) 60 60 0 5 Temperature + + + + Ionic strength + + + + High level (+) 240 85 10 20 Desorption time + + + +

Figure 4. Effect of solvent for each individual compound during the LD step.

dichloromethane, ethyl acetate, toluene, toluene/ACN 10:90, and toluene/ACN 20:80 were chosen. By covering a wide range of polarities, we tried to identify the solvent with the best capacity to remove the pesticides from the bar. Ethyl acetate, toluene, and dichloromethane were not good for desorption because they cause PDMS phase deterioration. The mixture of toluene/ACN 20:80 had a higher striping capacity for almost all the pesticides studied, while not offending the sorbent stability. The sonication implemented at this stage was tested and the best condition established was 15 min (Fig. 4). The use of home-made bars has the advantage of lowering the extraction cost allowing the evaluation of different solvents used in the LD, which may cause in many cases the PDMS phase deterioration. The amount of solvent in the desorption process was also investigated; at the beginning 200 lL of solvent was used, but when the amount of solvent was increased to

1.5 mL, evaporated to dryness and reconstituted in 50 lL a large improvement in the peak chromatographic area, as in Fig. 5, was observed. Possible carryover was removed by keeping the bar in the oven at 2508C for additional time (1 h) after the LD. During the chromatographic analysis the ions were selected by the inspection of the fragmentogram of each compound. The selected ions and respective retention time for the analytes investigated are described in Table 2.

3.3 Validation
To evaluate the developed bars some validation parameters were selected. The LOD obtained, based on a S/N of 3 was between 0.01 and 1.59 ng/g. The LOQ, based on a S/N of 10 was between 0.04 and 4.76 ng/g [50]. Precision was evaluated by the analysis of six replicates with two different bars in spiked samples at the concentration of 11 ng/
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Figure 5. A representative chromatogram obtained by HS-SBSE-GC-MS. 100 ppb spiked water sample with 13 OCPs (1, a HCH; 2, b HCH; 3, c HCH; 4, D HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, a endosulfan; 9, DDE; 10, dieldrin; 11, endrin; 12, DDD; and 13, DDT). Part (a) of the chromatogram shows the desorption in 200 lL of solvent while (b) shows the desorption in 1.5 mL of solvent, which is evaporated to dryness and reconstituted in 50 lL. In both cases, 2 lL of the final solution was injected into the GC-MS system in order to compare the areas obtained. The interrupted traces ( ) in the chromatograms are introduced to allow a quick visual comparison of the areas.

Table 2. Corresponding octanol water partitioning coefficients (log kO/W), selected ions (m/z) and respective retention time (tR) for the analytes investigated under the experimental conditions described in this study Pesticide a HCH b HCH c HCH D HCH Heptachlor Aldrin Heptachlor epoxide a Endosulfan DDE Dieldrin Endrin DDD DDT log kO/Wa) 4.26 3.68 4.26 3.68 5.86 6.75 4.56 3.50 6.00 5.45 5.45 5.87 6.79 m/z 183; 181; 109 183; 181; 109 183; 181; 109 183; 181; 109 100; 272; 274 66; 263; 220 353; 355; 351 195; 339; 341 246; 248; 176 79; 263; 279 263; 82; 81 235; 237; 165 235; 237; 165 tR (min) 16.15 17.01 17.33 18.14 20.73 22.82 25.51 28.47 30.33 30.88 32.99 34.59 39.05

Ions were selected by the inspection of the fragmentogram of each compound. a) From ref. [20].

g and measured by the variation coefficient, with the results obtained between 2.1 and 14.8%. In general the precision should be a 20% per level [51]. Linearity was evaluated by using the external standardization method with spiked samples at five concentration levels (at each concentration level, triplicate analyses were performed) with a linear range between 5 and 17 ng/g and r2 between 0.9602 and 1.000 (Table 3). The major goal of this paper is the (in-house) development and evaluation (using a validation process with spiked samples) of stir bars. In this research, the LOD and LOQ are limited by the injection and not by the extraction technique. There are specific injection devices for use with Stir Bar Sorptive Extraction, including: (i) large volume injection (LVI) when the analytes are desorbed from Stir Bar with solvent, the LVI can inject all solvents and all analytes extracted, increasing the LOQ. (ii) Thermal desorption (TD) when the analytes are desorbed from Stir Bar with temperature, all analytes are directed to be analyzed, increasing the LOQ. In this research, the

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Table 3. Values of RSD, regression coefficient (r2), LOD, LOQ, theoretical recovery for the studied compounds Pesticide a HCH b HCH c HCH D HCH Heptachlor Aldrin Heptachlor epoxide a Endosulfan DDE Dieldrin Endrin DDD DDT
a)

Precision 7.2 3.6 2.1 5.3 14.8 6.5 10.7 7.8 6.0 4.9 12.5 12.5 7.3

r2 1.0000 0.9993 0.9969 0.9971 0.9866 0.9748 0.9956 0.9994 0.9776 0.9990 0.9713 0.9602 0.9857

LOD (ng/g) 0.02 1.59 0.31 0.09 0.07 0.21 0.26 0.11 0.01 0.35 0.58 0.38 0.3

LOQ (ng/g) 0.07 4.76 0.93 0.28 0.23 0.63 0.77 0.33 0.04 1.1 1.73 1.13 0.9

Recovery (%)a) 97.7 91.8 97.7 99.8 99.9 100.0 98.8 88.1 100.0 99.8 99.8 99.9 100.0

Theoretical recovery calculated based on log kO/W of each pesticide.

analytes were desorbed from the Stir Bar with 1.5 mL of solvent, being injected, with a conventional chromatography syringe, only 2 lL into the column (splitless mode). The proposal of this research was the development and evaluation of sorptive stir bars and not the optimization of the LODs and LOQs, but is clear that it can be decreased by just using more appropriate injection devices. According to SBSE theory, the distribution coefficients of the analytes between PDMS and the water matrix (KPDMS/w) are strongly correlated with the corresponding kO/W. It is expected that nonpolar pesticides (log kO/W A 5) should have substantial affinity for the PDMS polymeric coating of the bars. Assuming a 15 mL water sample (Vw) and a bar coated with 35 lL (VSBSE) the phase ratio b could be calculated (b = Vw/VSBSE) and was established as 428.6. Using the phase ratio it was possible to calculate the theoretical recovery (Table 3). According to the literature [20] there is an agreement between the theoretical recovery and the experimental data, based on this and our results we suppose that the novel bar could achieve good recovery results under the optimized experimental conditions.

The authors would like to acknowledge FAPESP and CAPES for financial support to this research. The authors declared no conflict of interest.

5 References
[1] Kawaguchi, M., Ito, R., Saito, K., Nakazawa, H., J. Pharm. Biomed. Anal. 2006, 40, 500 508. [2] Huang, S. P., Huang, S. D., J. Chromatogr. A 2007, 1176, 19 25. [3] Pawliszyn, J., Arthur, C. L., Anal. Chem. 1990, 62, 2145 2148. [4] Queiroz, M. E. C., Lanas, F. M., LCGC North Am. 2004, 22, 970 980. [5] Takeshi, K., Lee, X., Sato, K., Suzuki, O., Anal. Chim. Acta 2003, 492, 49 67. [6] Queiroz, M. E. C., Valado, C. A. A., Farias, A., Carvalho, D., Lanas, F. M., J. Chromatogr. B 2003, 794, 337 342. [7] Queiroz, M. E. C., Silva, S. M., Carvalho, D., Lanas, F. M., J. Sep. Sci. 2002, 25, 91 95. [8] Queiroz, M. E. C., Silva, S. M., Carvalho, D., Lanas, F. M., J. Chromatogr. Sci. 2002, 40, 219 223. [9] Aresta, A., Cioffi, N., Palmisano, F., Zambonin, C. G., J. Agric. Food Chem. 2003, 51, 5232 5237. [10] Rodrigues, M. V. N., Reyes, F. G. R., Rehder, V. L. G., Rath, S., Chromatographia 2005, 61, 291 297. [11] Zeng, E. Y., Tsukada, D., Noblet, J. A., Peng, H., J. Chromatogr. B 2005, 1066, 165 175. [12] Albanis, A. T., Hela, D. G., Lambropoulou, D. A., Sakkas, V. A., Int. J. Environ. Anal. Chem. 2004, 84, 1079 1092. [13] Beltran, J., Lpez, F. J., Hernndez, F., J. Chromatogr. A 2000, 885, 389 404. [14] Blasco, C., Font, G., Manes, J., Pico, Y., Anal. Chem. 2003, 75, 3606 3615. [15] Baltussen, E., Sandra, P., David, F., Cramers, C., J. Microcol. Sep. 1999, 11, 737 747. [16] Lon, V., Alvarez, B., Cobollo, M., Muoz, S., Valor, I., J. Chromatogr. A 2003, 999, 91 101. [17] Pealver, A., Garcia, V., Pocurull, E., Borrull, F., Marc, R. M., J. Chromatogr. A 2003, 1007, 1 9. [18] Mothes, S., Popp, P., Wennrich, R., Chromatographia 2003, 57, S249 S252.

4 Concluding remarks
The present results show that the (in-house) developed bars used for HS sorptive extraction followed by LD and by GC coupled to MS offer excellent features for the extraction of OCP in water samples being cost-effectiveness once they do not require the use of expensive thermal desorption equipment. Moreover, the development of a technique to produce sorptive bars has as additional advantage, i. e., the possibility to use different materials and film thickness with specific characteristics adequate for different analyses.

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[19] Bicchi, C., Cordero, C., Rubiolo, P., Sandra, P., J. Sep. Sci. 2003, 26, 1650 1656. [20] Serdio, P., Nogueira, J. M. F., Anal. Chim. Acta 2004, 517, 21 32. [21] Popp, P., Bauer, C., Wennrich, L., Anal. Chim. Acta 2001, 436, 1 9. [22] Vercauteren, J., Peres, C., Devos, C., Sandra, P., Vanhaecke, F., Moens, L., Anal. Chem. 2001, 73, 1509 1514. [23] Kolahgar, B., Hoffmann, A., Heiden, A., J. Chromatogr. A 2002, 963, 225 230. [24] Sandra, P., Tienpont, B., David, F., J. Chromatogr. A 2003, 1000, 299 309. [25] Demyttenaere, J. C. R., Martinez, J. I. S., Verhe, R., Sandra, P., De Kimpe, N. J., J. Chromatogr. A 2003, 985, 221 232. [26] Hayasaka, Y., MacNamara, K., Baldock, G. A., Taylor, R. L., Pollnitz, A. P., Anal. Bioanal. Chem. 2003, 375, 948 955. [27] Bicchi, C., Iori, C., Rubiolo, P., Sandra, P., J. Agric. Food Chem. 2002, 50, 449 459. [28] Benijts, T., Vercammen, J., Dams, R., Tuan, H., Lambert, W., Sandra, P., J. Chromatogr. B 2001, 755, 137 142. [29] Tienpont, B., David, F., Bicchi, C., Sandra, P., J. Microcolumn Sep. 2000, 12, 577 584. [30] Bicchi, C., Cordero, C., Iori, C., Rubiolo, P., Sandra, P., J. High Resolut. Chromatogr. 2000, 23, 539 546. [31] Page, B. D., Lacroix, G. J., J. Chromatogr. 1997, 757, 173 182. [32] Ng, W. F., Teo, M. J. K., Lakso, H. A., Fresenius J. Anal. Chem. 1999, 363, 673 679. [33] Guan, F., Watanabe, K., Ishii, A., Seno, H., Kumazawa, T., Hattori, H., Suzuki, O., J. Chromatogr. B 1998, 71, 205 213. [34] Doong, R.-A., Liao, P.-L., J. Chromatogr. A 2001, 918, 177 188. [35] Li, H.-P., Li, G.-C., Jen, J.-F., J. Chromatogr. A 2003, 1012, 129 137. [36] Fernandez-Alvarez, M., Llompart, M., Lamas, J. P., Lores, M., Garcia-Jares, C., Cela, R., Dagnac, T., J. Chromatogr. A 2008, 1188, 154 163.

[37] Derouiche, A., Driss, M. R., Morizur, J.-P., Taphanel, M.-H., J. Chromatogr. A 2007, 1138, 231 243. [38] You, J., Lydy, M. J., Int. J. Environ. Anal. Chem. 2006, 86, 381 389. [39] Zhao, R. S., Wang, X., Fu, S., Yuan, J. P., Jiang, T., Xu, S. B., Anal. Bioanal. Chem. 2006, 384, 1584 1589. [40] Serdio, P., Nogueira, J. M. F., Anal. Bioanal. Chem. 2005, 382, 1141 1151. [41] Koester, C. J., Simonich, S. L., Esser, B. K., Anal. Chem. 2005, 75, 2813 2829. [42] Kuehl, R. O., Design of Experiments: Statistical Principles of Research Design and Analysis, Duxbury Press, Belmont 1994. [43] Montgomery, D. C., Design an Analysis of Experiments, Wiley & Sons, New York 1997. [44] Serdio, P., Cabral, M. S., Nogueira, J. M. F., J. Chromatogr. A 2007, 1141, 259 270. [45] Quintana, J. B., Rodil, R., Lorenzo, S. M., Lpez-Maha, P., PradaRodriguz, D., J.Chromatogr. A 2007, 1174, 27 39. [46] Bourdat-Deschamps, M., Daudin, J. J., Barriuso, E., J. Chromatogr. A 2007, 1167, 143 153. [47] Prieto, A., Zuloaga, O., Usobiaga, A., Etxebarria, N., Fernndez, L. A., J. Chromatogr. A 2007, 1174, 40 49. [48] Atkinson, A. C., Tobias, R. D., J. Chromatogr. A 2008, 1177, 1 11. [49] Carasek, E., Cudjoe, E., Pawliszyn, J., J. Chromatogr. A 2007, 1138, 10 17. [50] Clesceri, L. S., Greenberg, A. E., Eaton, A. D. (Eds.), Standard Methods for the Examination of Water and Wastewater, 20th Edn., American Public Health Association, Washington, DC, USA 1995. [51] European Commission, Directive 91/414/EEC, Official. J. Eur. Communities 1991, L 230, 1 26.

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