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    Optimization of parameters for the alcoholic-assisted dispersiveliquidliquid microextraction of estrogens in water

    Panteha Shakeri Zahra Mousavi Kiasari

    Mohammad Reza Hadjmohammadi

    Mohammad Hossein Fatemi

    Received: 7 July 2013 / Accepted: 30 December 2013

    Iranian Chemical Society 2014

    Abstract Extraction and determination of estrogens in

    water samples were performed using alcoholic-assisted

    dispersive liquidliquid microextraction (AA-DLLME)

    and high-performance liquid chromatography (UV/Vis

    detection). A PlackettBurman design and a central com-

    posite design were applied to evaluate the AA-DLLME

    procedure. The effect of six parameters on extraction

    efficiency was investigated. The factors studied were vol-

    ume of extraction and dispersive solvents, extraction time,

    pH, amount of salt and agitation rate. According to

    PlackettBurman design results, the effective parameters

    were volume of extraction solvent and pH. Next, a central

    composite design was applied to obtain optimal condition.

    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,

    86.7, respectively. The relative standard deviations for

    determination of estrogens in water were in the range of

    3.97.2 % (n = 3). The developed method was success-

    fully applied for the determination of estrogens in envi-

    ronmental water samples.

    Keywords Alcoholic-assisted dispersive liquidliquid

    microextraction Optimization Estrogens Experimentaldesign PlackettBurman design


    AA-DLLME Alcoholic-assisted dispersive liquidliquid


    EDCs Endocrine disrupting chemicals

    E1 Estrone

    E2 17b-estradiol

    DLLME Dispersive liquidliquid microextraction

    LLE Liquidliquid extraction

    SPE Solid-phase extraction

    CPE Cloud point extraction

    SBSE Stir bar sorptive extraction

    SPME Solid-phase microextraction

    PB PlackettBurman design

    CCF Central composite face-centered

    ER Extraction recovery

    EF Enrichment factor

    ANOVA Analysis of variance

    R2 Coefficient of determination


    The fact that some chemicals may disrupt the endocrine

    systems in humans and animals has received considerable

    attention in the scientific and public community. Such

    chemicals are widely referred to as endocrine disrupting

    chemicals (EDCs), and are on the agenda of many expert

    groups, steering committees and panels of governmental

    organizations, industries and academia throughout the

    world. Exposure to EDCs may have little effect on the

    exposed organism, but the offspring of that organism may

    suffer drastic repercussions [1]. Recently, there has been a

    growing worldwide concern on EDCs due to their high

    toxicity. Among the EDCs known to effect people, the

    P. Shakeri Z. Mousavi Kiasari M. R. Hadjmohammadi (&) M. H. Fatemi

    Faculty of Chemistry, University of Mazandaran, Babolsar, Iran

    e-mail: hadjmr@umz.ac.ir



    DOI 10.1007/s13738-013-0403-5

  • most important ones are the natural estrogens, estrone (E1)

    and 17b-estradiol (E2), which display higher estrogenic

    capacities and have thousand times higher biological

    potency than other compounds such as bisphenol A, al-

    kylphenols and nonylphenols [25]. Therefore, the pre-

    sence of E1 and E2 in aquatic environments will pose a

    serious threat to the local organisms and human health [6].

    The environmental concentrations for these estrogens

    are very low; therefore, a sensitive, selective and simple

    method requires monitoring them in water [7]. Before

    determination of these materials in water samples they

    require a pretreatment technique. Many different pretreat-

    ment techniques, such as liquidliquid extraction (LLE) [8,

    9], solid-phase extraction (SPE) [10, 11], solid-phase

    microextraction (SPME) [12], stir bar sorptive extraction

    (SBSE) [13] and cloud point extraction (CPE) [14] were

    used for the extraction of estrogens. Unfortunately, the

    traditional methods such as LLE and SPE require a large

    consumption of organic solvents, sample volume and are

    time consuming. Although SPME and SBSE are both sol-

    vent-free techniques, the fibers of SPME are fragile,

    expensive and have limited lifetime and sample carries

    over is the other problem of this technique. For SBSE, an

    additional desorption step is required when it couples with

    HPLC. CPE uses surfactants for extraction thus the choices

    of the surfactants often bring the nuisance to the analysis of

    analytes using GC and HPLC [1519]. Recently, a new

    microextraction method, named dispersive liquidliquid

    microextraction (DLLME), introduced by Assadi et al. [20]

    has been used as a powerful preconcentration technique for

    extraction of a variety of compounds including estrogens

    [2123]. The main disadvantage of the common DLLME

    technique is the use of chlorinated solvents as extraction

    solvent that are potentially toxic to humans and the envi-

    ronment. In addition, because the extraction solvent is

    incompatible with liquid chromatography (LC), DLLME

    extract cannot be injected directly to LC system for ana-

    lysis. On the other hand, in the determination of some

    important compounds, for example organochlorine pesti-

    cides using DLLME-GC-electron capture detector, chlori-

    nated extraction solvents have a very high solvent peak

    which covers some analytes peaks. To develop the appli-

    cability of the DLLME procedure, the alcoholic-assisted

    dispersive liquidliquid microextraction (AA-DLLME)

    method was introduced in our laboratory [24]. The basic

    criteria in AA-DLLME for selection of alcoholic solvents

    as extraction and dispersive solvents are their less toxicity

    and environmental greenness. In comparison of DLLME

    and AA-DLLME, the former needs higher volumes of

    dispersive solvent (in the mL range). Furthermore, the

    tedious procedure of evaporation of extraction solvent in

    DLLME, which may cause the loss of analyte, was elimi-

    nated in the AA-DLLME procedure and the extraction

    solvent can be directly injected into HPLC. Moreover, AA-

    DLLME method is environmentally greener than other

    DLLME procedures due to the use of alcoholic solvents

    [2527]. The main aim of the present work was to inves-

    tigate and optimize the extraction conditions of AA-

    DLLME procedure using PlackettBurman factorial design

    (PBD) and central composite face-centered (CCF) design.

    Then, the developed method was used for analysis of

    estrogens in water samples.


    Reagents and standards

    Estrone and 17b-estradiol were purchased from SigmaAldrich (St. Louis, MO, USA). 1-Octanol and 1-heptanol

    were purchased from Fluka (Buches, Switzerland). Etha-

    nol, methanol (HPLC-grade), acetonitrile (HPLC-grade),

    2-ethyl-1-hexanol, sodium chloride, sodium hydroxide and

    hydrochloric acid, were obtained from Merck (Darmstadt,

    Germany). Double distilled deionized water was produced

    by a Milli-Q system (Millipore, Bedford, MA, USA). Stock

    solutions of estrogens (500.0 mg L-1) were prepared in

    methanol and stored in the dark at 4 C. The workingsolutions were prepared daily by an appropriate dilution of

    the stock solution in water. All solutions were filtered

    through 0.45 lm membrane filters (Millipore, Bedford,MA) prior to use.


    The chromatographic separations were carried out on a 1525

    solvent delivery system and a model 2487 UV/Vis selective

    wavelength detector set at 280 nm, all from Waters (Mil-

    ford, MA, USA). The analytical isocratic RP-HPLC sepa-

    ration was performed on a C18 column (250 9 4.6 mm,

    5 lm) from Dr. Maisch (Beim Brueckle, Germany) at roomtemperature. Mobile phase was a mixture of acetonitrile:

    water (50:50, v/v), with flow rate of 1.0 ml min-1. The

    injection volume was 20 lL. A Hettich centrifuge modelUNIVERSAL 320 (Tuttlingen, Germany) was used to

    accelerate the phase separation. A Jenway model 3030 pH

    meter equipped with a combined glasscalomel electrode

    was employed for pH measurement. The magnetic stirrer

    used was MR 2002 (Heidolph, Germany). All statistical

    analyses were performed with Statgraphics 5.1.

    Alcoholic-assisted dispersive liquidliquid

    microextraction procedure

    For AA-DLLME, 10 ml of aqueous standard (pH 6)

    including the analytes (100 lg L-1) was poured into a



  • specially designed glass cell (Fig. 1) containing a magnetic

    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

    syringe (Reno, NV, USA) while solution was being stirred

    at 1,250 rpm. After the injection a cloudy solution was

    formed, and the extraction solvent was floated on the neck

    of glass cell. Afterward the cell was centrifuged for 10 min

    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 s


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