Journal of Chromatography A, 1190 (2008) 27–38

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Journal of Chromatography A

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Ultrasound-assisted emulsification–microextraction of emergent contaminantsand pesticides in environmental waters

Jorge Regueiroa, Maria Llomparta, Carmen Garcia-Jaresa,∗, Juan C. Garcia-Monteagudob, Rafael Celaa

a Departamento de Quimica Analitica, Nutricion y Bromatologia, Instituto de Investigacion y Analisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela15782, Spainb Departamento de Quimica Fisica, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain

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Article history:Received 18 October 2007Received in revised form 8 February 2008Accepted 29 February 2008Available online 5 March 2008

Keywords:Ultrasound-assistedemulsification–microextractionMicroextractionSynthetic musk fragrancesPhthalate estersLindaneFactorial experimental designWater analysis

a b s t r a c t

The analytical use of ultraefficiency in liquid–liquidimmiscible phases impliethe continuous phase, anon ultrasound-assisted emmass spectrometry (GC/Mesters and lindane in wateCompounds were extractroform and 10 mL sampleaccuracy (recovery = 78–1(LODs) were at the pg mL−

uitous phthalate esters. USother extraction techniqueing bottled, tap, river, mueffect has been found forconventional external caliother microextraction tec

1. Introduction

Sample preparation represents a major challenge and a veryimportant step in the development and application of an analyticalmethod. In general, this step consists of an extraction and precon-centration procedure of target compounds from a sample matrix.

Liquid–liquid extraction (LLE) [1,2] and solid-phase extraction(SPE) [3,4] are commonly used liquid sample pretreatment meth-ods. LLE is among the oldest and more widespread techniques forthe extraction of a wide range of organic pollutants from water sam-ples. Nevertheless, LLE is time-consuming, requires large amountsof organic solvents that are potentially toxic, and is difficult to auto-mate. SPE uses much less solvent than LLE but can be relativelyexpensive.

Over the last 10 years, with the developing interest in miniatur-ization in analytical chemistry with resultant solvent and samplesavings, some newer miniaturized approaches to liquid extrac-

∗ Corresponding author. Tel.: +34 981563100x14394; fax: +34 981595012.E-mail address: qncgj@usc.es (C. Garcia-Jares).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2008.02.091

d-generated emulsions has recently found a growing interest to improvection since they increase the speed of the mass transfer between the twos, dispersed droplets can act as efficient liquid–liquid microextractors in

r they can be readily separated by centrifugation. A novel method basedfication–microextraction (USAEME) and gas chromatography coupled tos been developed for the analysis of synthetic musk fragrances, phthalateples. Extraction conditions were optimized using a multivariate approach.ring 10 min in an acoustically emulsified media formed by 100 �L chlo-ichment factor = 100). The method performance was studied in terms oflinearity (R2 ≥ 0.9990) and repeatability (RSD ≤ 14%). Limits of detection

el for most of compounds, and at the sub-ng mL−1 level for the most ubiq-E is proposed as an efficient, fast, simple and non-expensive alternative toh as SPE, SPME and LPME for the analysis of environmental waters includ-al swimming pool, sewage and seaport water samples. Since no matrixf the water types analyzed, quantification could be carried out by using

on, thus allowing a higher throughput of the analysis in comparison withes based on equilibrium such as solid-phase microextraction.

© 2008 Elsevier B.V. All rights reserved.

tion have been reported. These approaches have resulted inmore efficient sample enrichment, faster sample preparation andlower solvent consumption. An attractive alternative pretreatmentmethod to the traditional techniques is solid-phase microextrac-tion (SPME) [5,6]. SPME is a solvent-free extraction techniquethat incorporates sample pretreatment, concentration and sam-ple introduction into a single procedure. But the extraction fiber isexpensive, fragile and has a limited lifetime, and in addition, sam-ple carry-over can be a problem [7]. Liquid–liquid microextraction(LLME) is a single-step extraction with a very high sample-to-solvent ratio which leads to a high enrichment factor of analytes. So,conventional LLME has been proposed in several US EPA methodsas an efficient alternative to LLE [8,9].

In the past few years, a novel liquid–liquid microextractionsystem, termed liquid-phase microextraction (LPME) or solventmicroextraction (SME), was developed [10,11]. This approach isbased on analyte partitioning between a drop of organic solvent(extractant phase) and the aqueous sample matrix. Different con-figurations of this technique have recently emerged, including staticLPME, dynamic LPME, continuous-flow LPME, headspace LPME(HS-LPME) and hollow fiber LPME [12–14]. This technique has

matog

28 J. Regueiro et al. / J. Chro

attracted increasing attention in recent years because of the sim-ple experimental setup, short analysis time and minimum useof solvent. However, several disadvantages such as instability ofmicrodrop and relative low precision are often encountered.

Very recently, a novel microextraction technique, dispersiveliquid–liquid microextraction (DLLME), based on dispersion of tinydroplets of the extraction liquid within the aqueous solution hasbeen developed [15]. It is based on a ternary component solventsystem like homogeneous liquid–liquid extraction (HLLE) [16] andcloud point extraction (CPE) [17]. The advantages of the DLLMEmethod are rapidity, low cost and high enrichment factors. Its maindrawbacks are the difficulty to automate and the necessity of usinga third component (disperser solvent), which usually decreases thepartition coefficient of analytes into the extractant solvent.

On the other hand, the application of ultrasonic radiation is apowerful aid in the acceleration of various steps of the analyti-cal process, therefore ultrasound assisted liquid–liquid extraction(USALLE) has been used as an alternative to conventional LLE byLuque de Castro’s research group [18,19], who also successfullyapplied ultrasound assisted emulsification (USAE) for the first timeto simultaneously determine polar and non-polar compounds insolid plant samples [20]. They demonstrated high extraction effi-ciency in a very short time using an emulsion of methanol/water inhexane formed in the presence of ultrasound radiation.

In a heterogeneous system of two immiscible liquid phases theeffect of ultrasound radiation is the concurrent result of severalpartial phenomena with complex interrelationships and depen-dent on a considerable number of variables [21–24]. Regardingliquid–liquid extraction, the main effects of ultrasounds can besummarized as follows: the fragmentation of one of the phases toform emulsions with submicron droplet size [25] that enormouslyextend the contact surface between both liquids; the inverse effect(coalescence) occurring in certain conditions [22,26] as a result ofLangevin or Rayleigh pressures; the homogenization of the exter-nal phase by the action of acoustic flows [22,27]; the incrementof temperature [22,23]; and the momentary and localized strongincrements of pressure and temperature in the proximity of thecavitational collapses [25,26] that, when originated near to theliquid–liquid interface, may selectively affect the exchange of com-ponents without scarcely altering the whole of the emulsion.

The application of a miniaturized approach to this techniqueby using a microvolume of extracting organic phase, providesthe advantages of both DLLME [15] and USALLE [18] and somemore, mainly derived from the low concentration of inner phasedrops: decrease of the coalescence effect [26,28], decrease of the

radiation absorption and the resultant warming [23], and theacoustic flow facilitation [29] with the result of an homogeniza-tion speed increase. The consequence is a very efficient and fastanalyte extraction. After mass transfer, the two phases can be read-ily separated by centrifugation. In this way, ultrasound-assistedemulsification–microextraction (USAEME) can be employed as asimple and efficient extraction and preconcentration procedure fororganic compounds in aqueous samples.

Phthalic acid esters are a group of chemical compounds thatare mainly used as plasticizers and some of them also as carri-ers or solvents for synthetic musks fragrances in many personalcare products (PCPs). Significant migration into the environment isdemonstrated during their production, manufacture, use and dis-posal [30,31]. Synthetic musks are fragrance additives used in awide range of consumer products. Nowadays, polycyclic musks likegalaxolide and tonalide form part of most fragrance formulationsfor household and cosmetic products. Nitromusks are currentlypresent in about 5% of the products that were not reformulated, andalthough nitromusks are being phased out in Europe, completion isexpected in 2008 [32,33]. Since musk compounds are continuouslyintroduced into the environment mainly via urban wastewater

r. A 1190 (2008) 27–38

effluents [34,35], their environmental persistence associated withtheir lipophilicity makes their routine monitoring in wastewatersand other environmental samples still necessary today. Gamma-hexachlorocyclohexane (�-HCH), commonly known as lindane, isa pesticide that has been used in a broad range of applicationsincluding agriculture, horticulture and forestry. Because of its ubiq-uity, lindane is still found in environmental samples [36]. Thedevelopment of simple, sensitive and reliable analytical methodsto analyze these compounds in different water samples is hencenecessary.

The aim of the present work is to propose a novel method basedon USAEME and gas chromatography coupled to mass spectrome-try (GC/MS) for the analysis of synthetic musk fragrances, phthalateesters and lindane in water samples. To the best of our knowledge,this paper describes the first application of ultrasound-assistedemulsification microextraction for the determination of organiccompounds in water samples without the addition of an emulsifier.Optimization of the extraction conditions is achieved using a multi-factorial experimental design approach. The method performanceis studied in terms of accuracy, linearity, repeatability and limits ofdetection (LODs). To demonstrate the applicability of the proposedmethod, several types of water samples including plastic-bottledwater, tap water, river water, municipal swimming pool water, seaharbour water and sewage waters, are analyzed.

2. Experimental

2.1. Reagents and materials

The musk compounds, 6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H)-indanon (DPMI, cashmeran) and 4-acetyl-1,1-dimethyl-6-tert-butylindan (ADBI, celestolide) were kindly supplied by Ventos(Cornella de Llobregat, Barcelona, Spain). 1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(�)-2-benzopyran (HHCB,galaxolide), 7-acetyl-1,1,3,4,4,6-hexamethyltetraline (AHTN,tonalide), 6-acetyl-1,1,2,3,3,5-hexamethylindane (AHMI, phan-tolide) and 5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane (ATII,traseolide) were supplied by Promochem Iberia (Barcelona,Spain). The nitromusk fragrances 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenezene (musk xylene), 4,6-dinitro-1,1,3,3,5-penta-methylindane (musk moskene) and 4-tert-butyl-3,5-dinitro-2,6-dimethylacetophenone (musk ketone) were obtained as100 �g mL−1 solutions in acetonitrile from Riedel de Haen (Seelze,Germany). �-Hexachlorocyclohexane (lindane), 1000 �g mL−1 in

methanol was supplied by Supelco (Bellefonte, P.A., USA).

Dimethyl phthalate (>98%) and diethyl phthalate (>98%) werepurchased from Fluka (Buchs, Switzerland), di-n-butyl phthalate(>98%) and di-2-ethylhexyl phthalate (>99%) were from Sigma(St. Louis, MO, USA) and benzyl butyl phthalate (97.2%) anddi-n-octyl phthalate (99.7%) were supplied by Riedel-de Haen(Seelze-Hannover, Germany) and Supelco (Bellefonte, PA, USA),respectively.

The surrogate standard PCB-166 (2,3,4,4′,5,6-hexachloro-biphenyl) and the internal standard PCB-195 (2,2′,3,3′,4,4′,5,6-octachlorobiphenyl) were purchased as 10 �g mL−1 solutions inisooctane from Dr. Ehrenstorfer (Augsburg, Germany).

Table 1 shows the CAS registry numbers, molecular weights,octanol–water partition coefficients (log Kow) and chemical struc-tures of the target compounds.

Isooctane, ethyl acetate, acetone, chloroform and sodium chlo-ride were provided by Merck (Darmstadt, Germany). Chloroben-zene and 1,1,1-trichloroethane were obtained from Sigma (St. Louis,MO, USA), whereas carbon tetrachloride was purchased from Fluka(Buchs, Switzerland). All solvents and reagents were analyticalgrade.

J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38 29

Table 1Molecular weight, octanol–water partition coefficient (log Kow) and structure of the studied compounds

Compound CAS number MW log Kow Structure

DMP 131–11–3 194.2 1.61

DEP 84–66–2 222.2 2.54

DBP 84–74–2 278.3 4.27

BBP 85–68–7 312.4 4.70

DEHP 117–81–7 390.6 7.73

DOP 117–84–0 390.6 7.73

Cashmeran 33704–61–9 206.3 4.9

Celestolide 13171–00–1 244.4 6.6

Phantolide 15323–35–0 244.4 6.7

Traseolide 68140–48–7 258.4 8.1

30 J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38

Table 1 (Continued )

Compound CAS number MW log Kow Structure

Galaxolide 1222–05–5 258.4 5.9

Tonalide 1506–02–1 258.4 5.7

97.3

78.3

94.3

90.8

Musk xylene 81–15–2 2

Musk moskene 116–66–5 2

Musk ketone 81–14–1 2

Lindane 58–89–9 2

Individual stock solutions of each compound were preparedin acetone and a standard mixture solution of all target com-pounds was prepared in acetone at a final concentration of about200 �g mL−1. Different standard working solutions were obtainedby appropriate dilution and stored in amber colored vials at −20 ◦C.

Different real water samples were analyzed: plastic-bottledwater, tap water, river water, municipal swimming pool water, seaharbour water, and influent and effluent waters from an STP cor-

responding to a population of approximately 100,000 inhabitantslocated in the North-West of Spain. Samples were filtrated througha 0.45 �m Millipore HA membrane filter (Billerica, MA, USA) andstored in glass bottles at 4 ◦C.

2.2. Ultrasound-assisted emulsification–microextraction(USAEME)

Aliquots of 10 mL sample were placed in 15 mL conical-bottomglass centrifuge tubes, which were previously cleaned accordingto the procedure described later. Prior to extraction, 5 ng of PCB-166 (in acetone) were added to each sample as surrogate standard.Under final optimized conditions, 100 �L of chloroform containing5 ng of PCB-195 (internal standard) were added as extractant sol-vent. The tube was then immersed into an ultrasonic water bathSelecta Ultrasounds (Barcelona, Spain) in such a way that the levelof both liquids (bath and sample) was the same. Extractions wereperformed at 40 kHz of ultrasound frequency and 100 W of powerfor 10 min at 25 ± 3 ◦C at the beginning of every experiment. Asa result, oil-in-water (O/W) emulsions of chloroform (dispersedphase) in water (continuous phase) were formed. Emulsions were

4.8

5.8

4.3

3.9

then disrupted by centrifugation at 5000 rpm for 3 min and theorganic phase was sedimented at the bottom of the conical tube.Chloroform was removed by using a 100 �L Hamilton syringe (Reno,NV, USA) and transferred to a 100 �L glass insert located in a 1.8 mLgas chromatography vial. The thus obtained extracts were stored at−20 ◦C until analysis by GC/MS.

2.3. Gas chromatography–mass spectrometry

The GC/MS analysis were performed using a Varian 3800 gaschromatograph (Varian Chromatography Systems, Walnut Creek,CA, USA) coupled to an ion trap mass detector Varian Saturn 2000.The system was operated by Saturn GC/MS Workstation v5.4 soft-ware.

Separation was carried out on a VF-5ms FactorFour capillarycolumn (30 m × 0.25 mm i.d., 0.25 �m film thickness), also fromVarian. Helium (purity 99.999%) was employed as carrier gas ata constant column flow of 0.8 mL min−1. The GC oven temperaturewas programmed from 50 ◦C (hold 2 min) to 190 ◦C at 20 ◦C min−1

and then until 270 ◦C (hold 5 min) at 5 ◦C min−1 (total analysis time:30 min). Injections (1 �L) were done in the splitless mode (hold2 min), using an injector temperature of 280 ◦C.

The ion trap mass spectrometer was operated in the electronimpact ionization (EI) positive mode (+70 eV). Instrumental param-eters for ionization were as follows: filament emission current15 �A, filament/multiplier delay 8 min, electron multiplier poten-tial 1600 V, axial modulation amplitude 4 V, multiplier offset 100 Vand perfluorotributylamine (PFTBA) as calibration gas. The massrange was scanned in the full scan mode from 80 to 450 m/z. The

J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38 31

Table 2Retention times and selected ions for the analysis of the target compounds

Compounds Retention time (min)

DMP 9.15Cashmeran 9.56DEP 10.17Celestolide 11.29Phantolide 11.72Lindane 11.84Traseolide 12.67Galaxolide 12.74Musk xylene 12.84Tonalide 12.85Musk moskene 13.12DBP 13.90Musk ketone 14.28BBP 19.34PCB-166 (SS) 19.98DEHP 22.13PCB-195 (IS) 24.07DOP 24.77

SS = surrogate standard. IS = internal standard.

scan time for data acquisition was set at 0.8 s, with 3 microscans s−1.Trap, manifold and transfer line temperatures were maintained at250, 120 and 300 ◦C, respectively. The target compounds were pos-itively identified by comparison of their mass spectra and retention

times to those of standard solutions.

3. Results and discussion

3.1. Performance of the GC/MS analysis

First experiments were conducted to achieve good chromato-graphic separation of the target compounds. The mass spectrumfor each compound was obtained in the experimental conditions,and the most adequate ions for quantification were selected. Reten-tion times at the optimized chromatographic conditions, as well asthe identification and quantification ions are shown in Table 2.

Linearity was tested for most of the target compounds between1 ng mL−1 and 500 ng mL−1 by injecting standards prepared in chlo-roform at seven different concentrations in duplicate. Linear rangesfor the rest of compounds are indicated in Table 3. Determinationcoefficients (R2) between 0.9990 and 0.9998 were obtained for allcompounds (Table 3). Relative standard deviations for five consec-utive injections of 100 ng mL−1 standard solution ranged from 1%to 7%. Instrumental detection limits (IDLs) estimated for a signal-to-noise of 3 were in the pg mL−1 level for all compounds, with the

Table 3Performance of the GC/MS analysis

Compounds Linear range(ng mL−1)

R2 RSD (%, n = 5)/100 ng mL−1

IDL(ng mL−1)

DMP 10–500 0.9991 4 2.73Cashmeran 1–500 0.9998 6 0.14DEP 5–500 0.9992 3 0.54Celestolide 1–500 0.9992 7 0.22Phantolide 1–500 0.9991 6 0.21Lindane 5–500 0.9994 2 1.00Traseolide 5–500 0.9991 2 0.50Galaxolide 1–500 0.9992 1 0.23Musk xylene 1–500 0.9990 1 0.16Tonalide 1–500 0.9991 3 0.17Musk moskene 1–500 0.9993 4 0.07DBP 5–500 0.9992 5 0.55Musk ketone 1–500 0.9990 3 0.08BBP 10–500 0.9991 5 2.88DEHP 10–500 0.9990 3 2.94DOP 30–500 0.9996 3 8.11

IDL = instrumental detection limit.

Quantification ions (m/z) Identification ions (m/z)

163 164,194191 206, 135149 177, 222229 244, 173229 244, 187181, 183, 219 217, 109215 258, 173243 213, 258282 265, 297243 258, 159263 278, 264149 223, 205279 294, 128149 206, 238358, 360, 362 290, 288149 167, 279429, 430, 431 358, 360149 167, 279

exception of DMP, BBP, DEHP and DOP whose IDLs were in the lowng mL−1 level (Table 3).

3.2. Preliminary experiments

It is well known that the most important problem concerningphthalate analysis is the risk of contamination, resulting in falsepositive results and over-estimated concentrations. The sources ofcontamination can be present in any step of the analytical proce-dure. Special care was taken to avoid the contact of reagents andsolutions with plastic materials. Laboratory glassware was washedprior to use with ultrapure water and dried at 300 ◦C. This materialwas stored in aluminum foil to avoid adsorption of phthalates fromthe air. To check the presence of phthalates in the chromatographicsystem (in the inlet and the gas supply system), blank runs of thechromatograph and direct injections of solvents were made. Thepresence of phthalate esters was not detected.

Besides, due to the occurrence of musk fragrances as ingredientsof all kinds of cleansing products and cosmetics, the risk of samplecontamination when they are manipulated in the laboratory is notnegligible, so it is advisable to extreme precautions to avoid sourcesof interference in the laboratory environment.

Regarding the USAEME process, the selection of a suitableextractant is limited by several characteristics that are necessary

for emulsification in the presence of ultrasonic radiation. Some ofthese characteristics are a higher density than water and low watersolubility. Furthermore, selected solvent must be compatible withthe separation and detection technique and, therefore a good gaschromatographic behavior is another desirable characteristic.

Most of the heavy solvents are halogenated compounds; severalof them were initially tested in order to evaluate their emulsifi-cation capacity and the possible presence of interferences duringmass spectrometry detection. Dichloromethane (CH2Cl2), car-bon tetrachloride (CCl4), chloroform (CHCl3), tetrachloroethylene(C2Cl4) and 1,1,1-trichloroethane (C2H3Cl3) were initially consid-ered as possible extracting solvents. Their main physical propertiesare shown in Table 4.

Aliquots of 5 mL of ultrapure water and 100 �L of every solventwere US irradiated for 10 min and emulsification was observed inall cases with the exception of CH2Cl2. The higher water solubil-ity and volatility of dichloromethane might result in no emulsionformation, so this solvent was ruled out for further optimization.

Organic phases were then separated by centrifugation andinjected into the GC/MS system. Obtained chromatograms werechecked for interferences and best results were observed for CHCl3

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32 J. Regueiro et al. / J. Chro

Table 4Physicochemical properties of the solvents studied as possible extractants

Solvents Density 20 ◦C (g mL−1) Vapour pressure 20 ◦C (kPa)

CH2Cl2 1.33 47.4CHCl3 1.48 21.2CCl4 1.59 12.2C2H3Cl3 1.34 13.3C2Cl4 1.62 1.90

and C2H3Cl3. Therefore, these solvents were used for the next stepsof method development.

In all the experiments, PCB-195 was used as internal standard.In this way, possible problems resulting from lack of repeatabilityin the volume of the sedimented phase are minimized.

3.3. Optimization of USAEME process: screening factorial design

The influence of the main variables potentially affecting the effi-ciency of ultrasound-assisted emulsification–microextraction wasevaluated by using a multifactorial screening design. The study con-sisted of a mixed level fraction 3 × 23−1 design plus 4 centerpoints,involving 16 randomized experiments and allowing 5 degrees offreedom to estimate the experimental error. This design has reso-lution V, which means that it is capable of evaluating all main effectsand all two-factor interactions. Numerical analysis of data resultingfrom the experimental design was made with the statistical soft-ware package Statgraphics-Plus v5.1 (Manugistics, Rockville, MD,USA). Factorial design optimization was performed using aliquotsof a tap water sample spiked with the analytes at a concentrationof 10 ng mL−1.

Extraction time is usually an important factor in the most ofextraction procedures. The effect of extraction time was examinedat three levels between 1 min and 10 min.

The selection of an appropriate extractant is an importantparameter for all LLE-based processes. As commented in prelimi-

nary experiments, two organic solvents were previously selectedattending to both their capability to be dispersed in water andtheir good GC/MS behaviour. These solvents, chloroform andtrichloroethane, were tested in the experimental design in anattempt to achieve the highest extraction efficiency for the targetcompounds.

The influence of the volume ratio between the two liquid phaseswas also considered in the study. The volume of organic solventwas kept constant at 100 �L whereas the aqueous phase volumeranged from 5 mL to 10 mL. Thus, the phase volume ratio (Va/Vo) wasstudied at values of 50 and 100, respectively. Obtained responsesfor every compound were divided by the used volume in order toobtain a relative measure of the effect of this variable.

The salting-out effect has been frequently used in LLE, SPME andLPME. Generally, addition of salt can decrease the solubility of ana-lytes in the aqueous phase and promote the transfer of the analytestowards the organic phase. Therefore, the concentration of sodiumchloride in the aqueous solution was the last factor evaluated attwo levels: 0% (no addition) and 20% (m/v).

In brief, four variables were screened in this design, namely:extraction time, extractant, phase volume ratio and concentration

Table 5Factors and levels selected for the screening design

Factors Key Lower level Intermed

Extraction time A 1 5.5Extractant B CHCl3 –Volume ratio (Va/Vo) C 50 –NaCl concentration D 0 –

Va = aqueous phase volume (mL). Vo = organic phase volume (mL).

r. A 1190 (2008) 27–38

ater solubility 20 ◦C (g mL−1) log Kow Dipole moment 20 ◦C (D)

.013 1.25 1.14

.008 1.97 1.15

.0008 2.64 0

.0005 2.49 1.78

.00015 2.86 1.32

of sodium chloride. The upper and lower values attributed to eachfactor were selected from the experience gathered in preliminaryexperiments. The levels given to each factor, as well as the identifi-cation key, are summarized in Table 5.

Numerical analysis of the results leads to the Pareto charts formain factors and two-factor interactions, which are shown in Fig. 1for several selected compounds. The length of each bar is propor-tional to the absolute value of its associated standardized effect.The standardized effect is obtained by dividing the estimated effectof each factor or interaction by its standard error. Vertical line inthe graphs represents the statistically significant bound at the 95%confidence level.

As can be seen, the most important factor with statistical signif-icance for all studied compounds was the extractant. Furthermore,this factor was the only significant one for several compounds suchas DMP, DEP, DBP and musk xylene.

Extraction time presented a significant effect for polycyclicmusk fragrances, musk moskene, and the phthalates DEHP andDOP. For these phthalates, its influence is also manifested by theexistence of a significant AA interaction. This second order factorrepresents the quadratic effect of this factor.

Concentration of sodium chloride was a significant factor for allpolycyclic musk fragrances, musk ketone, lindane and the higherphthalates (BBP, DEHP, and DOP).

Phase volume ratio had no statistical significance for any ofthe target compounds, which means that extraction efficiency is

not affected by the volumes in the studied range. In this way, thehigher ratio (100) should be selected to improve the sensitivity ofthe method.

Regarding the two-factor interactions, time–extractant interac-tion (AB) was significant for cashmeran, celestolide and galaxolide,whereas the interaction between extracting solvent and volumeratio (BC) showed significance for phantolide, celestolide and galax-olide. The interactions between time and volume (AC) and betweenvolume and concentration of sodium chloride (CD) were statisti-cally significant for celestolide and galaxolide.

Fig. 2 shows the main effect plots for several representative com-pounds. The plots for the rest of the compounds are not includedin this figure since all of them were very similar, which will enableto select common extraction conditions for all target compounds.This kind of plots shows the main effects with a line drawn betweenthe low and the high level of the corresponding factors. The lengthof the lines is proportional to the effect magnitude of each factor inthe extraction process, and the sign of the slope indicates the levelof the factor that produces the highest response.

As commented above, extractant was the most important fac-tor for all compounds and its influence is clearly appreciated in the

iate level Upper level Units Continuous

10 min YesC2H3Cl3 – No100 – Yes20 % Yes

J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38 33

selec

Fig. 1. Pareto charts for

plots. Higher extraction efficiency was observed when chloroformwas used as extractant. A possible explanation for this effect mightbe related to the lower dipole moment of this solvent (1.15 D) incomparison with trichloroethane (1.78 D), allowing a better inter-action between solvent molecules and non-polar compounds. Onthe other hand, a lower interfacial tension between water andchloroform would enable a higher cavitation under ultrasound irra-diation and hence, a higher efficiency in emulsion formation.

With regards to extraction time, it can be observed that higherresponses were obtained in the upper level of this factor, i.e.10 min. As known, fast rates of mass transport are supposed duringliquid–liquid processes in acoustically emulsified media. Therefore,very short times should be required in comparison with conven-tional liquid–liquid extraction techniques. Nevertheless, the useof an ultrasonic bath rather than an ultrasonic probe has beenreported to produce a lower rate of emulsification [37]. It mightexplain the need of using the upper level of extraction time since ahigher time is necessary for obtaining a smaller droplet size, whichenables a more efficient emulsification and extraction. On the otherhand, an extraction time of 10 min can be considered short enoughfor an exhaustive extraction technique, and much more attend-

ted target compounds.

ing to the low time consumption during the rest of the proceduresteps.

Concentration of sodium chloride was a significant factor forthose compounds presenting higher octanol–water partition coef-ficients (log Kow) such as polycyclic musk fragrances and higherphthalate esters (see Table 1). All of them were negatively affectedby the presence of the salt during the extraction process.

Attending to the salting-out effect, influence of a salt addi-tion should be favourable since it decreases the water solubilityof analytes enabling a higher mass transfer towards the organicphase. Nevertheless, viscosity also plays an important role in thiskind of extraction. So, ultrasounds can be absorbed by the viscousresistance of the solution and dispersed as calorific energy. As aconsequence, the organic phase is not able to be dispersed in sofine droplets and therefore, efficiency of emulsion formation canbe drastically reduced.

In this way, the effect of salt addition can be considered as theresult of two major competitive effects, namely: salting-out effectand viscous resistance effect. As can be expected, salting-out effectshould be more important for the most polar compounds and botheffects might be counteracted. Therefore, salt addition would be

34 J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38

repre

Fig. 2. Main effects plots for

a non-significant factor for these compounds. For the least polar

analytes, salting-out effect is lower and it might be less impor-tant than viscous resistance effect, which would lead to a negativesignificant effect of salt addition. It should be pointed out that anegative influence of salt addition on the extraction yield has alsobeen observed in other microextraction techniques such as SPMEand LPME [38,39].

Interaction graphs of the two-factor interactions relevant forcelestolide and galaxolide are depicted in Fig. 3.

Concerning the interaction between time and extractant (AB), itcan be observed that a higher response is obtained in one minutewhen chloroform is used. At the upper level of time, responses forthese musk compounds are very similar using both extractants.Nevertheless, the lines do not cross in the studied ranges, so higherresponses are always achieved with chloroform.

Regarding the interaction between extractant and volume ratio(BC), it is shown that when chloroform is used, a ratio of 100 (10 mLof aqueous phase) leads to a more efficient extraction, which is inconcordance with conclusions extracted from main effects plots.

The analysis of the graph for the interaction between time andvolume ratio (AC), indicates that a ratio of 50 is preferred whenthe extraction time is 1 min. Nevertheless, a higher response for

sentative target compounds.

celestolide is reached at a ratio of 100 when the sample is extracted

during 10 min.

Interaction between volume ratio and sodium chloride concen-tration (CD) indicates that no addition of salt is highly preferred forgalaxolide extraction when a ratio of 100 is selected.

In view of the results of the experimental design, theselected conditions for the ultrasound-assisted emulsification–microextraction of the target compounds from water samples wereas follows: an extraction time of 10 min, chloroform as extractant,a phase volume ratio of 100 and no addition of sodium chloride.Therefore, compounds were extracted during 10 min in an acousti-cally emulsified media formed with 100 �L of chloroform and 10 mLof a water sample.

3.4. Study of method performance

Analytical quality parameters were measured in order to assessthe performance of the USAEME method.

Recoveries were evaluated using a tap water sample spiked atconcentrations of 0.1 ng mL−1 and 1 ng mL−1, respectively. The thusobtained values ranged from 91% to 114% at 0.1 ng mL−1 level andfrom 87% to 113% at 1 ng mL−1 level (Table 6).

J. Regueiro et al. / J. Chromatog

Fig. 3. Two-factor interactions graphs relevant for celestolide and galaxolide.

Precision of the experimental procedure was also evaluated atboth concentration levels by calculating the relative standard devi-ation (RSD) of three replicates. These results are also shown inTable 6. Values ranged from 5% for musk moskene to 12% for DMPand from 3% for musk xylene to 11% for DEHP, at the low and highconcentration levels, respectively.

To evaluate the performance of the method in terms of detec-tion limits, it is necessary, or at least convenient, to have waterfree of analytes to establish the background of the method. Nev-ertheless, one of the most important problems in the analysis ofphthalates in water samples is the detection of these compoundsin the samples used as blanks. Phthalates have been detected inpurified water commonly used in laboratories, including water dis-

Table 6Recovery, repeatability, limits of detection and limits of quantification of the proposed US

Compounds Recovery (%) Repeatability

0.1 ng mL−1 1 ng mL−1 0.1 ng mL−1

DMP 95 107 12Cashmeran 97 112 10DEP n.a. 113 n.a.Celestolide 96 100 8Phantolide 110 95 7Lindane 103 109 6Traseolide 92 87 10Galaxolide 114 91 8Musk xylene 108 108 4Tonalide 105 94 6Musk moskene 112 106 5DBP n.a. 110 n.a.Musk ketone 91 106 9BBP 97 109 5DEHP n.a. 94 n.a.DOP n.a. 110 n.a.

LOD = limit of detection. LOQ = limit of quantification. n.a. = not available, addition level <L

r. A 1190 (2008) 27–38 35

tilled in a glass distillation apparatus and ultrapure Milli-Q water.These blank signals forced the limits of detection achieved, mainlyfor DBP and DEHP, the most ubiquitous phthalate esters. In thepresent study, blank USAEM analyses were initially carried outwith Milli-Q water, and the presence of phthalates was detectedbeing DBP and DEHP the compounds found at the highest level.Hence, it is difficult to accurately assign the origin of the detectedphthalates. It may be attributed to the presence of low levels ofthese compounds in the ultrapure water or due to contaminationduring the analytical procedural stages. In spite of these resultsand because of the unavailability of perfect water blanks, ultrapurewater was adopted for estimation of detection limits (LOD = blanksignal + 3SD) taking into account the measured background levels.The thus estimated LODs are shown in Table 6. Values lower than10 pg mL−1 were obtained for all musk fragrances with the onlyexception of cashmeran (29.0 pg mL−1). Lindane and the phtha-lates DMP, BBP and DEP presented LODs lower than 50 pg mL−1,whereas values for DBP, DEHP and DOP were higher. These esti-mates maybe considered as conservative for DEP, DBP and DEHP.If the levels of phthalates found in blank sample analyses mightbe attributed to real sample contamination instead of process con-tamination, the estimated LODs for DEP, DBP and DEHP would beconsiderably lower.

The aim of the present work is developing a methodology

suitable for the analysis of the target compounds in differ-ent kinds of water samples including bottled mineral water,tap water, river water, swimming pool water, harbour seawa-ter and STP influent and effluent waters. Among them, influentand effluent waters are expected to present the most com-plex matrices, so they were initially selected to study possiblematrix effects. Samples were spiked with the target compoundsat 2 ng mL−1 and analyzed by the proposed method. As can beseen in Table 7, recoveries ranged from 83% to 114% (RSD = 4–12%) for the effluent sample and from 81% to 113% (RSD = 5–14%)for the influent sample, so no significant matrix effects were found.Recoveries were also evaluated in swimming pool water and ina harbour seawater sample (addition level of 1 ng mL−1), obtain-ing values between 82% and 103% (RSD = 1–13%) and between 78%and 91% (RSD = 1–11%), respectively (Table 7). USAEME can be con-sidered as an exhaustive microextraction technique and therefore,in the absence of matrix effects, quantification can be performedby external calibration using standards prepared in chloroform.In this way, and despite a lower sensitivity for most of the targetcompounds in comparison with other microextraction techniquesbased on equilibrium such as SPME [30,33,40], procedural sim-

AEME method

(RSD, % n = 3) LOD (pg mL−1) LOQ (pg mL−1)

1 ng mL−1

10 28 945 29 97

11 46 1539 9.6 326 7.4 259 21 718 8.3 287 7.0 233 10 346 6.0 206 9.4 31

10 125 4155 9.2 315 29 97

11 133 4428 98 326

OQ.

36 J. Regueiro et al. / J. Chromatogr. A 1190 (2008) 27–38

Table 7Recovery and repeatability of the proposed USAEME method for complex matrix water samples

Compounds Recovery (%) Repeatability (RSD, % n = 3)

STPeffluenta

STPinfluenta

Swimmingpool waterb

Harbourseawaterb

Effluent Influent Swimmingpool water

Harbourseawater

DMP 83 95 82 79 8 11 9 9Cashmeran 92 110 93 84 4 13 7 2DEP 106 102 94 82 5 6 9 7Celestolide 96 113 90 84 7 5 12 6Phantolide 94 100 93 91 5 6 9 7Lindane 98 101 95 83 2 11 9 3Traseolide 108 89 93 82 7 14 10 2Galaxolide 88 113 86 90 6 12 11 5Musk xylene 109 91 97 85 5 6 4 2Tonalide 91 112 89 80 4 12 9 1Musk moskene 114 81 103 87 5 8 5 2DBP 87 102 91 78 7 12 6 2Musk ketone 111 85 92 82 12 11 9 1BBP 106 81 88 79 7 14 13 11DEHP 94 86 84 83 5 7 8 7DOP 107 89 91 82 7 10 1 2

a Addition level of 2 ng mL−1.b Addition level of 1 ng mL−1.

Fig. 4. GC/MS extracted ion chromatograms for an STP effluent water sample spiked at 2 ng mL−1 of every target compound.

matog

ng poo

J. Regueiro et al. / J. Chro

Table 8Target compounds found in different types of water

Compounds Concentration (pg mL−1)

Bottled water Tap water River water Swimmi

DMP n.d. n.d. 158 ± 3 n.d.DEP 245 ± 23 195 ± 3 261 ± 26 345 ± 31Celestolide n.d. n.d. n.d. n.d.Phantolide n.d. n.d. n.d. n.d.Traseolide n.d. n.d. n.d. n.d.Galaxolide n.d. n.d. n.d. 274 ± 30Tonalide n.d. n.d. n.d. 119 ± 11DBP n.d. n.q. n.q. 663 ± 37

Musk ketone n.d. n.d. n.d. n.d.DEHP n.q. n.q. n.q. n.q.

n.d. = not detected (<LOD). n.q. = not quantified (<LOQ).

plicity and higher throughput are obtained. In Fig. 4, the GC/MSextracted ion chromatograms for a spiked effluent water sampleare displayed.

3.5. Application to real samples

The proposed method was then applied to the analysis of sev-eral non-spiked water samples. Analyses were performed at leastin triplicate and results of the target compounds found in sev-eral types of water samples are shown in Table 8. DEP, DBP andDEHP were the compounds present to a greater extent, espe-cially in the wastewater samples. Among them, DEHP was foundin higher concentrations, which could be expected since it is themost used plasticizer. DBP was detected in all analyzed samplesexcepting bottled water and its concentration was higher thanLOQ in the swimming pool water, in the seaport water and inSTP influent water. DMP was detected only in river water at a

Fig. 5. GC/MS extracted ion chromatograms for

r. A 1190 (2008) 27–38 37

l water Harbour seawater STP effluent water STP influent water

n.d. n.d. n.d.340 ± 23 428 ± 34 679 ± 63n.d. n.d. n.q.n.d. 31 ± 2 n.d.n.d. n.d. n.q.n.d. 718 ± 44 2893 ± 289n.d. 99 ± 7 334 ± 30425 ± 9 n.q. 617 ± 34

n.d. n.d. 113 ± 6n.q. 550 ± 30 1901 ± 204

level of 158 pg mL−1. Synthetic musk fragrances were found inthe swimming pool water and in sewage waters. Galaxolide andtonalide were the dominant fragrances at concentrations rangingfrom the pg mL−1 level to the low ng mL−1 level. Celestolide andtraseolide were detected in the influent sample, although theirconcentration levels were under LOQ. The presence of phantolideat 31 pg mL−1 in the effluent of the treatment plant cannot berelated to its absence in the influent water since both samples arenot correlated. Musk ketone was also determined in the influentwater at a concentration of 113 pg mL−1, which demonstrates thatnitromusk compounds are still present in PCPs and hence, theyshould be monitored in water samples. The rest of synthetic muskcompounds as well as lindane were not found in any of the sam-ples.

Extracted ion chromatograms for a non-spiked STP influ-ent water sample are shown in Fig. 5 (see concentrations inTable 8).

a non-spiked STP influent water sample.

matog

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[

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ultrasonidos, Mir, Moscow, 1990.

38 J. Regueiro et al. / J. Chro

4. Conclusions

A method based on ultrasound-assisted emulsification–microextraction (USAEME) coupled to GC/MS has been developedfor the analysis of synthetic musk fragrances, phthalate esters andlindane in water samples. USAEME is proposed as an efficient,simple, rapid and non-expensive alternative to other extractiontechniques such as SPE, SPME and LPME. Besides, it is environ-mentally friendly because of the low organic solvent consumptionand easy to automate. Using the optimized conditions establishedafter a multivariate study of the USAEME process, good recover-ies were obtained for all compounds, even in complex wastewatersamples. Method precision was satisfactory and detection limitsat the picogram per millilitre level were achieved for most of thetarget compounds, and at the sub-nanogram per millilitre level forthe most ubiquitous phthalate esters. The proposed method wasthen applied to the analysis of several real water samples includ-ing tap water, plastic-bottled water, swimming pool water, harbourseawater and wastewaters. Since no matrix effects were observedeven in the most complex samples, quantification could be easilyperformed by external calibration using standards in chloroform,allowing high throughput of the analysis and procedural simplicity

in comparison with other non-exhaustive microextraction tech-niques like SPME. Phthalate esters, mainly DEP and DEHP, weredetected in all analyzed samples, whereas synthetic fragranceswere found in the swimming pool water and in the wastewatersamples, being galaxolide and tonalide the dominant compounds.

Acknowledgements

This research was supported by FEDER funds and projectsCTQ2006-03334 (CICYT, Ministerio de Ciencia y Tecnologia, Spain),PGIDT06PXI3237039PR and PGIDIT05RAG50302PR (Xunta de Gali-cia). J. Regueiro would like to acknowledge his FPU doctoral grantto Ministerio de Ciencia y Tecnologia.

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