Optimization of parameters for the alcoholic-assisted dispersive liquid–liquid microextraction of estrogens in water

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<ul><li><p>ORIGINAL PAPER</p><p>Optimization of parameters for the alcoholic-assisted dispersiveliquidliquid microextraction of estrogens in water</p><p>Panteha Shakeri Zahra Mousavi Kiasari </p><p>Mohammad Reza Hadjmohammadi </p><p>Mohammad Hossein Fatemi</p><p>Received: 7 July 2013 / Accepted: 30 December 2013</p><p> Iranian Chemical Society 2014</p><p>Abstract Extraction and determination of estrogens in</p><p>water samples were performed using alcoholic-assisted</p><p>dispersive liquidliquid microextraction (AA-DLLME)</p><p>and high-performance liquid chromatography (UV/Vis</p><p>detection). A PlackettBurman design and a central com-</p><p>posite design were applied to evaluate the AA-DLLME</p><p>procedure. The effect of six parameters on extraction</p><p>efficiency was investigated. The factors studied were vol-</p><p>ume of extraction and dispersive solvents, extraction time,</p><p>pH, amount of salt and agitation rate. According to</p><p>PlackettBurman design results, the effective parameters</p><p>were volume of extraction solvent and pH. Next, a central</p><p>composite design was applied to obtain optimal condition.</p><p>The optimized conditions were obtained at 220 lL 1-oct-anol as extraction solvent, 700 lL ethanol as dispersivesolvent, pH 6 and 200 lL sample volume. Linearity wasobserved in the range of 1500 lg L-1 for E2 and0.1100 lg L-1 for E1. Limits of detection were0.1 lg L-1 for E2 and 0.01 lg L-1 for E1. The enrichmentfactors and extraction recoveries were 42.2, 46.4 and 80.4,</p><p>86.7, respectively. The relative standard deviations for</p><p>determination of estrogens in water were in the range of</p><p>3.97.2 % (n = 3). The developed method was success-</p><p>fully applied for the determination of estrogens in envi-</p><p>ronmental water samples.</p><p>Keywords Alcoholic-assisted dispersive liquidliquid</p><p>microextraction Optimization Estrogens Experimentaldesign PlackettBurman design</p><p>Abbreviations</p><p>AA-DLLME Alcoholic-assisted dispersive liquidliquid</p><p>microextraction</p><p>EDCs Endocrine disrupting chemicals</p><p>E1 Estrone</p><p>E2 17b-estradiol</p><p>DLLME Dispersive liquidliquid microextraction</p><p>LLE Liquidliquid extraction</p><p>SPE Solid-phase extraction</p><p>CPE Cloud point extraction</p><p>SBSE Stir bar sorptive extraction</p><p>SPME Solid-phase microextraction</p><p>PB PlackettBurman design</p><p>CCF Central composite face-centered</p><p>ER Extraction recovery</p><p>EF Enrichment factor</p><p>ANOVA Analysis of variance</p><p>R2 Coefficient of determination</p><p>Introduction</p><p>The fact that some chemicals may disrupt the endocrine</p><p>systems in humans and animals has received considerable</p><p>attention in the scientific and public community. Such</p><p>chemicals are widely referred to as endocrine disrupting</p><p>chemicals (EDCs), and are on the agenda of many expert</p><p>groups, steering committees and panels of governmental</p><p>organizations, industries and academia throughout the</p><p>world. Exposure to EDCs may have little effect on the</p><p>exposed organism, but the offspring of that organism may</p><p>suffer drastic repercussions [1]. Recently, there has been a</p><p>growing worldwide concern on EDCs due to their high</p><p>toxicity. Among the EDCs known to effect people, the</p><p>P. Shakeri Z. Mousavi Kiasari M. R. Hadjmohammadi (&amp;) M. H. Fatemi</p><p>Faculty of Chemistry, University of Mazandaran, Babolsar, Iran</p><p>e-mail: hadjmr@umz.ac.ir</p><p>123</p><p>J IRAN CHEM SOC</p><p>DOI 10.1007/s13738-013-0403-5</p></li><li><p>most important ones are the natural estrogens, estrone (E1)</p><p>and 17b-estradiol (E2), which display higher estrogenic</p><p>capacities and have thousand times higher biological</p><p>potency than other compounds such as bisphenol A, al-</p><p>kylphenols and nonylphenols [25]. Therefore, the pre-</p><p>sence of E1 and E2 in aquatic environments will pose a</p><p>serious threat to the local organisms and human health [6].</p><p>The environmental concentrations for these estrogens</p><p>are very low; therefore, a sensitive, selective and simple</p><p>method requires monitoring them in water [7]. Before</p><p>determination of these materials in water samples they</p><p>require a pretreatment technique. Many different pretreat-</p><p>ment techniques, such as liquidliquid extraction (LLE) [8,</p><p>9], solid-phase extraction (SPE) [10, 11], solid-phase</p><p>microextraction (SPME) [12], stir bar sorptive extraction</p><p>(SBSE) [13] and cloud point extraction (CPE) [14] were</p><p>used for the extraction of estrogens. Unfortunately, the</p><p>traditional methods such as LLE and SPE require a large</p><p>consumption of organic solvents, sample volume and are</p><p>time consuming. Although SPME and SBSE are both sol-</p><p>vent-free techniques, the fibers of SPME are fragile,</p><p>expensive and have limited lifetime and sample carries</p><p>over is the other problem of this technique. For SBSE, an</p><p>additional desorption step is required when it couples with</p><p>HPLC. CPE uses surfactants for extraction thus the choices</p><p>of the surfactants often bring the nuisance to the analysis of</p><p>analytes using GC and HPLC [1519]. Recently, a new</p><p>microextraction method, named dispersive liquidliquid</p><p>microextraction (DLLME), introduced by Assadi et al. [20]</p><p>has been used as a powerful preconcentration technique for</p><p>extraction of a variety of compounds including estrogens</p><p>[2123]. The main disadvantage of the common DLLME</p><p>technique is the use of chlorinated solvents as extraction</p><p>solvent that are potentially toxic to humans and the envi-</p><p>ronment. In addition, because the extraction solvent is</p><p>incompatible with liquid chromatography (LC), DLLME</p><p>extract cannot be injected directly to LC system for ana-</p><p>lysis. On the other hand, in the determination of some</p><p>important compounds, for example organochlorine pesti-</p><p>cides using DLLME-GC-electron capture detector, chlori-</p><p>nated extraction solvents have a very high solvent peak</p><p>which covers some analytes peaks. To develop the appli-</p><p>cability of the DLLME procedure, the alcoholic-assisted</p><p>dispersive liquidliquid microextraction (AA-DLLME)</p><p>method was introduced in our laboratory [24]. The basic</p><p>criteria in AA-DLLME for selection of alcoholic solvents</p><p>as extraction and dispersive solvents are their less toxicity</p><p>and environmental greenness. In comparison of DLLME</p><p>and AA-DLLME, the former needs higher volumes of</p><p>dispersive solvent (in the mL range). Furthermore, the</p><p>tedious procedure of evaporation of extraction solvent in</p><p>DLLME, which may cause the loss of analyte, was elimi-</p><p>nated in the AA-DLLME procedure and the extraction</p><p>solvent can be directly injected into HPLC. Moreover, AA-</p><p>DLLME method is environmentally greener than other</p><p>DLLME procedures due to the use of alcoholic solvents</p><p>[2527]. The main aim of the present work was to inves-</p><p>tigate and optimize the extraction conditions of AA-</p><p>DLLME procedure using PlackettBurman factorial design</p><p>(PBD) and central composite face-centered (CCF) design.</p><p>Then, the developed method was used for analysis of</p><p>estrogens in water samples.</p><p>Experimental</p><p>Reagents and standards</p><p>Estrone and 17b-estradiol were purchased from SigmaAldrich (St. Louis, MO, USA). 1-Octanol and 1-heptanol</p><p>were purchased from Fluka (Buches, Switzerland). Etha-</p><p>nol, methanol (HPLC-grade), acetonitrile (HPLC-grade),</p><p>2-ethyl-1-hexanol, sodium chloride, sodium hydroxide and</p><p>hydrochloric acid, were obtained from Merck (Darmstadt,</p><p>Germany). Double distilled deionized water was produced</p><p>by a Milli-Q system (Millipore, Bedford, MA, USA). Stock</p><p>solutions of estrogens (500.0 mg L-1) were prepared in</p><p>methanol and stored in the dark at 4 C. The workingsolutions were prepared daily by an appropriate dilution of</p><p>the stock solution in water. All solutions were filtered</p><p>through 0.45 lm membrane filters (Millipore, Bedford,MA) prior to use.</p><p>Instrumentation</p><p>The chromatographic separations were carried out on a 1525</p><p>solvent delivery system and a model 2487 UV/Vis selective</p><p>wavelength detector set at 280 nm, all from Waters (Mil-</p><p>ford, MA, USA). The analytical isocratic RP-HPLC sepa-</p><p>ration was performed on a C18 column (250 9 4.6 mm,</p><p>5 lm) from Dr. Maisch (Beim Brueckle, Germany) at roomtemperature. Mobile phase was a mixture of acetonitrile:</p><p>water (50:50, v/v), with flow rate of 1.0 ml min-1. The</p><p>injection volume was 20 lL. A Hettich centrifuge modelUNIVERSAL 320 (Tuttlingen, Germany) was used to</p><p>accelerate the phase separation. A Jenway model 3030 pH</p><p>meter equipped with a combined glasscalomel electrode</p><p>was employed for pH measurement. The magnetic stirrer</p><p>used was MR 2002 (Heidolph, Germany). All statistical</p><p>analyses were performed with Statgraphics 5.1.</p><p>Alcoholic-assisted dispersive liquidliquid</p><p>microextraction procedure</p><p>For AA-DLLME, 10 ml of aqueous standard (pH 6)</p><p>including the analytes (100 lg L-1) was poured into a</p><p>J IRAN CHEM SOC</p><p>123</p></li><li><p>specially designed glass cell (Fig. 1) containing a magnetic</p><p>stirring bar. A mixture of extraction solvent (220 lL,1-octanol) and disperser solvent (700 lL, ethanol) wasrapidly injected into the sample solution by a Hamilton</p><p>syringe (Reno, NV, USA) while solution was being stirred</p><p>at 1,250 rpm. After the injection a cloudy solution was</p><p>formed, and the extraction solvent was floated on the neck</p><p>of glass cell. Afterward the cell was centrifuged for 10 min</p><p>at 3,000 rpm and a 100 lL Hamilton syringe was used toremove the extracted layer and 30 lL of this phase wasinjected into the HPLC system for quantification.</p><p>Calculation of enrichment factor, extraction recovery</p><p>and relative recovery</p><p>The enrichment factor (EF) during the AA-DLLME was</p><p>calculated according to the following equation:</p><p>EF Cf=Caq 1The Cf is the final concentration of analyte in the</p><p>floating phase, and Caq is the initial analyte concentration</p><p>within the sample.</p><p>Recovery (R) was calculated according to the following</p><p>equation:</p><p>ER nf</p><p>naq 100% Vf</p><p>Vaq </p><p>EF 100% Vf</p><p>Vaq</p><p> Cf</p><p>Caq</p><p> 100% 2where nf and naq are the number of moles of analyte finally</p><p>collected in the extraction solvent and the number of moles</p><p>of analyte originally present in the sample, respectively. In</p><p>the above equation, Vf is the volume of floating extraction</p><p>solvent and Vaq is the volume of sample. Vf was determined</p><p>by bending the glass cell and gathering the floating organic</p><p>solvent by a micro liter syringe (conditions were kept</p><p>constant, so the same sample volume was obtained).</p><p>The relative recovery (RR %) calculated from the fol-</p><p>lowing equation:</p><p>RR % Cfound Creal =Cadded 100 3where Cfound, Creal and Cadded are the concentration of</p><p>analyte after addition of known amount of standard to real</p><p>sample, the concentration of analyte in real sample and the</p><p>concentration of standard added to the real sample,</p><p>respectively.</p><p>Results and discussion</p><p>Selection of disperser and extracting solvents</p><p>To achieve good recovery for AA-DLLME of estrogens,</p><p>the selection of an appropriate mixture of extraction and</p><p>disperser solvents is very important. The extraction solvent</p><p>should have some properties to extract the analytes effi-</p><p>ciently such as lower density than water, low solubility in</p><p>water, extraction capability of interested compound and</p><p>good chromatographic behavior. In this work, three alco-</p><p>holic solvents including 2-ethyl-1-hexanol (density</p><p>0.834 g mL-1), 1-octanol (density 0.824 g mL-1) and</p><p>1-heptanol (density 0.819 g mL-1) were used as extraction</p><p>solvents. Disperser solvent should be miscible with both</p><p>water and extraction solvent and produce very fine droplet</p><p>of extraction solvent, when mixture of extraction and dis-</p><p>perser solvent was rapidly injected into the sample.</p><p>Methanol and ethanol, which have this ability, were</p><p>applied. All combinations of extraction and disperser sol-</p><p>vents were examined for finding the optimum solvents.</p><p>During this procedure other AA-DLLME factors were</p><p>maintained constant (150 lL of extraction solvent, 500 lLof disperser solvent, stirring rate of 500 rpm, 10 % salt in</p><p>sample solution, pH 7 and 5 min extraction time). The</p><p>obtained results indicated that maximum extraction effi-</p><p>ciency was achieved using 1-octanol and ethanol as</p><p>extraction and disperser solvents, respectively.</p><p>Factors screening</p><p>Screening design includes examining different factors for</p><p>the main effects to reduce the number of factors. A par-</p><p>ticular type of such designs is PBD [28]. This design is</p><p>very useful for preliminary studies or in initial optimization</p><p>steps. In PBD the interactions can be completely ignored,</p><p>so the main effects are calculated with a reduced number ofFig. 1 Schematic figure for container of AA-DLLME</p><p>J IRAN CHEM SOC</p><p>123</p></li><li><p>experiments. Based on the preliminary experiments, it was</p><p>suggested that six factors including volume of extracting</p><p>and disperser solvents, amount of salt in sample solution,</p><p>pH, extraction time and stirring rate can affect the AA-</p><p>DLLME efficiency. Based on the PBD, each factor was</p><p>examined at two levels: -1 for low level and ?1 for high.</p><p>Table 1 indicates the levels of six studied factors in this</p><p>work together with design matrix of PBD design. As can be</p><p>seen six assigned variables were screened in 12 experi-</p><p>mental runs and the recovery was taken as experimental</p><p>response (Table 1). The Statgraphics 5.1 software was used</p><p>to analyze the experimental results. The analysis of vari-</p><p>ance (ANOVA) method was used to evaluate main effects</p><p>of parameters. The normalized results of the experimental</p><p>design were evaluated at a 5 % of significance and ana-</p><p>lyzed by Standardized Pareto chart (Fig. 2). The vertical</p><p>line on the plot judges the effects that are statistically</p><p>significant (p \ 0.05). The bars, extending beyond theline, correspond to the effects that are statistically, sig-</p><p>nificant at the 95 % confidence level. According to these</p><p>results, pH and extraction solvent volume were selected</p><p>as the important factors in extraction of estrogens by</p><p>AA-DLLME method. To optimize experimental condi-</p><p>tions, a CCF design was performed by these factors.</p><p>Other factors including disperser solvent volume,</p><p>extraction time and stirring rate were considered as</p><p>insignificant factors in the studied range, therefore, their</p><p>levels were kept constant and were determined based on</p><p>their sign on Pareto chart.</p><p>Optimization of AA-DLLME conditions</p><p>To investigate and validate process parameters affecting</p><p>the extraction of estrogens and exact optimization of the</p><p>extraction condition, the three levels, CCF was applied.</p><p>The total number of experiments (N) is determined by the</p><p>following equation</p><p>N 2f 2f N0 4In this equation f is the number of factors and N0 is the</p><p>number of replicates at central point. The resulted design</p><p>had four factorial points, four star points and four center</p><p>points. The examined levels of the factors and the design</p><p>matrix are given in Table 2. The resulting 12 experiments</p><p>were carried out randomly, using 10 mL of spiked water</p><p>samples, containing 100 lg L-1 of analytes and using</p><p>Table 1 The experimental variables, levels, design matrix andresponse of the PlackettBurman design</p><p>Variables (Symbols) Low High</p><p>Volume of extraction solvent</p><p>(A) (lL)100 300</p><p>Volume of dispersion solvent</p><p>(B) (lL)500 700</p><p>Amount of salt (C) (% w/v) 0 10</p><p>pH (D) 3 12</p><p>Extraction time (E) (min) 0 10</p><p>Stirring rate (F) (rpm) 100 1,250</p><p>No. Parameters ER %</p><p>A B C D E F</p><p>1 -1 1 1 -1 1 -1 57</p><p>2 1 1 1 -1 1 1 86</p><p>3 1 1 -1 1 -1 -1 77</p><p>4 -1 1 1 1 -1 1 55</p><p>5 -1 -1 -1 -1 -1 -1 73</p><p>6 -1 -1 -1 1 1 1 24</p><p>7 1 -1 -1 -1 1 1 90</p><p>8 1 -1 1 1 -1 1 70</p><p>9 -1 1 -1 -1 -1 1 64</p><p>10 1 -1 1 -1 -1 -1 65</p><p>11 1 1 -1 1 1 -1 49</p><p>12 -1 -1 1...</p></li></ul>