Ultrasound-assisted emulsification microextraction followed by gas chromatography–mass spectrometry and gas chromatography–tandem mass spectrometry for the analysis of UV filters in water

Microchemical Journal 124 (2016) 530–539

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Ultrasound-assisted emulsification microextraction followed by gas chromatography–mass spectrometry and gas chromatography–tandem mass spectrometry for the analysis of UV filters in water Marlene Vila a, J. Pablo Lamas a, Carmen Garcia-Jares a, Thierry Dagnac b, Maria Llompart a,⁎ a b

Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, Campus Vida, University of Santiago de Compostela, E-15782, Santiago de Compostela, Spain Agricultural and Agronomic Research Centre (INGACAL-CIAM), Unit of Organic Contaminants, Apartado 10, 15080 A Coruña, Spain

a r t i c l e

i n f o

Article history: Received 27 July 2015 Received in revised form 25 September 2015 Accepted 25 September 2015 Available online 9 October 2015 Keywords: Ultrasound-assisted emulsification microextraction GC–MS/MS Personal care products (PCPs) Emerging pollutants Water samples UV-filters

a b s t r a c t A methodology based on ultrasound-assisted emulsification microextraction (USAEME) has been developed for the simultaneous analysis of ten UV filters (2-ethylhexyl methoxycinnamate (2EHMC), 4- methylbencylidene camphor (4MBC), benzyl salicylate (BS), ethylhexyl dimethyl PABA (EHPABA), ethylhexyl salicylate (EHS), etocrylene (Eto), homosalate (HMS), isoamyl methoxycinnamate (IAMC), menthyl anthranilate (MA) and octocrylene (OCR)) in different water samples employing two detection modes, gas chromatography–mass spectrometry (GC–MS), and gas chromatography–tandem mass spectrometry (GC–MS/MS). The extraction parameters such as the extraction solvent, the temperature and the time of extraction, and the addition of salt were optimized by means of experimental design tools. Selected conditions were 100 μL of chloroform, 10 mL of sample, 2 g of salt (NaCl) and 5 min of extraction. Good linearity (R2 N 0.9910), quantitative recoveries (N 90% for most of compounds) and satisfactory precision (RSD b 10% in most cases) were achieved under the optimal conditions. The methodology was successfully applied to the analysis of different types of water samples including seawater, spas, rivers, swimming pools, and aquaparks, in which most of the target compounds were found at concentrations sometimes over the part per million. Since no significant matrix effect has been found for any of the water types analyzed, quantification could be carried out by using conventional external calibration, thereby allowing a high analytical throughput and a valuable protocol simplification. © 2015 Elsevier B.V. All rights reserved.

1. Introduction UV filters are “substances which are exclusively or mainly intended to protect the skin against certain UV radiation by absorbing, reflecting or scattering UV radiation”[1]. Nowadays, there is a great awareness of how important it is to protect the skin from the UV radiation to prevent the occurrence of cancer. For this reason, the use of sunscreens is increasing not only in specific solar product range but also in daily creams, lip balms, etc. Moreover, these compounds are also found in waters of rivers, lakes, pools and wastewater among others because they can enter the environment through the bath, swimming, shower, etc. In fact, UV filters are already classified as environmental emerging contaminants. Furthermore, it is important to emphasize that many of these compounds are lipophilic and therefore they can bioaccumulate and biomagnify through the food chain, and they may also induce estrogenic [2–4] and antithyroid [5] effects. The increased use of cosmetics and, therefore, the greater occurrence of such compounds in water, along with their consideration as emerging contaminants have made

⁎ Corresponding author. Tel.: +34 881814225. E-mail address: [email protected] (M. Llompart).

http://dx.doi.org/10.1016/j.microc.2015.09.023 0026-265X/© 2015 Elsevier B.V. All rights reserved.

their study in water and in other environmental compartments a priority issue in advanced countries. These compounds have a variety of structures and can be classified into different families: benzophenone derivatives, p-aminobenzoic acid and its derivatives, salicylates, cinnamates, camphor derivatives, triazine derivatives, derivatives benzotriazole, benzimidazole derivatives and others. For this reason, it is complex to integrate all these substances in a single analysis. According to the review of Pedrouzo et al. [6], the most frequently UV filters found in water samples are 4-methylbenzylidene camphor (4MBC), 2-ethylhexyl methoxycinnamate (2EHMC), isoamyl methoxycinnamate (IAMC), octocrylene (OCR), 2-phenylbenzimidazole-5-sulfonic acid (PBSA), octyl dimethyl PABA (EHPABA) and some benzophenones. Concerning sample preparation, the general treatment of water samples is based on solid phase extraction (SPE). Regarding the UV filter extraction from water samples, SPE technique is the one preferred by the authors, as it can be concluded based on the high number of works where it is employed [6]. The cartridges more frequently utilized to carry out the extraction were Oasis HLB, which contain a universal polymeric reversed-phase sorbent [7,8]. Other cartridges such as C18 [9,10], Strata X [11] and Oasis MCX [6] were also used. The amount of sample utilized in these cases was between 100 and 1000 mL and the

M. Vila et al. / Microchemical Journal 124 (2016) 530–539

volume of extractant (normally ethylacetate/dichloromethane (1:1, v/v) for GC analysis or methanol for LC analysis) was between 3 and 30 mL. After elution, the extracts are concentrated by evaporation. Overall, the large amount of sample and the additional concentration step make the method tedious. Other types of sample preparation such as stir bar sorptive extraction (SBSE) [12,13] and solid-phase microextraction (SPME) [14] were utilized to a lesser extent with the same finality. Few publications show the use of other microextraction techniques such as membrane assisted liquid–liquid extraction (MALLE) [15], single drop microextraction (SDME) [16], hollow fiber-based liquid phase microextraction (HF-LPME) [17] and microextraction by packed sorbent (MEPS) [18]. Dispersive liquid–liquid microextraction (DLLME) [19,20] is another technique employed to extract UV filters from water samples that allows to use a little amount of extractant, reducing the consumption of solvent and obtaining a less environmental hazardous method. Other varieties of this last mentioned technique based on the aid of vortex to perform the emulsification such as vortex-assisted dispersive liquid–liquid microextraction (VADLLME) [21] or vortex-assisted emulsification microextraction (VAEME) [22] were also employed to extract UV filters from water samples. Some authors employed the power of ultrasound instead of the vortex to get the emulsification, through ultrasound-assisted dispersive liquid–liquid microextraction (USA-DLLME) [23,24] and, in several studies, using ionic liquids [25,26]. Recent reviews dealing with the employ of both vortex-assisted and ultrasounds-assisted extraction techniques indicate that its use in LLME continues to expand, being an excellent option for the efficient and rapid extraction of several types of analytes from different samples, as shown in the big amount of works where they were employed [27,28]. The use of ultrasound-assisted emulsification–microextraction (USAEME) appears as a good alternative method to carry out the extraction of sunscreens from water samples. This procedure proposed by Regueiro et al. is based on the emulsification of a microvolume of organic extractant in an aqueous sample by ultrasound radiation, and further separation of both liquid phases by centrifugation. The application of ultrasonic radiation accelerates the mass-transfer process between two immiscible phases, which, together with the large surface of contact between both phases, leads to an increase of the extraction efficiency in a short time (few minutes). In addition, since it is a miniaturized technique, the organic solvent consumption is minimum and, therefore, offers an environmental friendly technique. Thus, ultrasound-assisted emulsification–microextraction (USAEME) can be employed as a simple and efficient extraction and preconcentration procedure for organic compounds in aqueous samples. Regueiro et al. applied this technique to determine preservatives [29] and musk fragrances [30] in water samples. USAEME was also employed to extract polycyclic aromatic hydrocarbons [31], polybrominated flame retardants [32], polychlorinated biphenyls [33], pesticides [34,35], and allergen fragrances [36], among others. Only a previous work by Ge et al.[37] has addressed the use of this technique with ionic liquids as extracting solvent to analyze 4MBC and three benzophenones. The determination of the UV filters is commonly performed with gas or liquid chromatography coupled to mass or tandem mass spectrometry [6–15,18–20,38]. It is well known that this kind of detectors provides lower LODs (ng L−1) [8,20] than LC with UV detection (LC–UV), only reaching the low μg L−1 level [37,39].Sensitive techniques are required for the analysis of these compounds in water because they are supposed to be present at trace levels. The aim of the present work was to optimize, validate and put into practice a simple methodology based on ultrasound-assisted emulsification–microextraction followed by gas chromatography–mass spectrometry (USAEME–GC–MS) and gas chromatography–tandem mass spectrometry (USAEME–GC–MS/MS), for the simultaneous analysis of different classes of UV filters in water samples of different nature. This was the first time that USAEME has been employed to extract the target analytes from water samples (excluding 4MBC, analyzed). A multifactor

531

categorical design was carried out to simultaneously evaluate the main experimental factors affecting USAEME such as the extracting solvent, the addition of salt, the temperature and the time of extraction. The method performance was assessed in terms of accuracy, linearity, repeatability and limits of detection (LODs). To demonstrate the applicability of the proposed method, several types of water samples including swimming pool water, aquaparks, seawater, river water and spa water were analyzed.

2. Experimental 2.1. Standards, reagents and materials 2-Ethylhexyl methoxycinnamate (2EHMC; 98.5%) and 2,4,6trichlorobiphenyl (PCB 30) were supplied by Ehrenstorfer (Augsburg, Germany). 4-Methylbencylidene camphor (4MBC; 99.8%) was obtained from Alfa Aesar (Karlsruhe, Germany). Benzyl salicylate (BS; N99%) and menthyl anthranilate (MA; 99.9%) were acquired from Fluka (Saint Louis, MO, USA). Octocrylene (OCR; 99.1%), ethylhexyl dimethyl PABA (EHPABA; 98%), ethylhexyl salicylate (EHS; N99%) and homosalate (HMS; 99.9%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Isoamyl methoxycinnamate (IAMC; 96%) and etocrylene (Eto; 99.7%) were acquired from TCI (Tokyo, Japan). Acetone and methanol were provided by Merck (Darmstadt, Germany). Ethyl acetate and trichloroethane were acquired from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Carbon tetrachloride was purchased from Normasolv (Barcelona, Spain). Chloroform was supplied by Merck (Darmstadt, Germany). All solvents and reagents were of analytical grade. Individual stock solutions of each compound were prepared in acetone, methanol or ethyl acetate. Mixtures in acetone were prepared to spike water samples (when needed). Stock and working solutions were stored in a freezer at −20 °C protected from light. The samples employed were Milli-Q water, tap water, seawater, spa water, swimming pool water, aquaparks and river water. Water samples were collected in 14 mL plastic conical-bottom tubes provided with two spatulas of sodium thiosulfate (100 mg approximately), and stored in the dark at4 °C until their analysis.

2.2. USAEME procedure Aliquots of 10 mL sample were placed in 15 mL conical-bottom glass centrifuge tubes. Then, a pair of spatulas of sodium thiosulfate (~ 100 mg) was incorporated to prevent oxidation of the compounds and their reaction with chlorine, which can be present in some water samples like spas or swimming pools. Under final optimized conditions, 2 g of sodium chloride was included. Afterwards, 100 μL of chloroform containing 10 ng of PCB-30 (internal standard) was added as extracting solvent. This volume of extractant is an appropriate quantity since a minor volume would make the operability and the realization of replicates of injection difficult. The tube was then immersed into an ultrasonic water bath Raypa® model UCI 150 (Barcelona, Spain) in such a way that the level of both liquids (bath and sample) was the same. Extractions were performed at 35 kHz of ultrasound frequency for 5 min at 25 °C at the beginning of every experiment. As a result, oilin-water (O/W) emulsions of chloroform (dispersed phase) in water (continuous phase) were formed. Emulsions were then disrupted by centrifugation (Orto Alresa, Digicen 21) at 3500 rpm (relative centrifugal force (RCF): 1986 g) for 10 min and the organic phase was sedimented at the bottom of the conical tube. Chloroform was removed by using a 100 μL Hamilton syringe (Bonaduz, Switzerland) and transferred to a 100 μL glass insert located in a 1.8 mL gas chromatography vial. The thus obtained extracts were stored at − 20 °C until analysis by GC–MS or GC–MS/MS.

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M. Vila et al. / Microchemical Journal 124 (2016) 530–539

2.3. GC–MS and GC–MS/MS analysis The GC–MS analysis was performed using an Agilent 7890A (GC)– Agilent 5975C inert MSD with triple axis detector and an Agilent 7693 autosampler from Agilent Technologies (Palo Alto, CA, USA). The temperatures of the transfer line, the quadrupole and the ion source were set at 290, 150 and 230 °C, respectively. The system was operated by Agilent MSD ChemStation E.02.01.1177 software. Separation was carried out on a cross-linked 5%-phenyl/95%dimethylpolysiloxane Zebron ZB-5 capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) obtained from Phenomenex (Torrance, CA, USA). Helium (purity 99.999%) was employed as carrier gas at a constant column flow of 1.0 mL min−1. The GC oven temperature was programmed from 100 °C (held 1 min) to 290 °C (held 2.4 min) at 25 °C min−1. Pulsed splitless mode was used for injection (30 psi, held 1.2 min). After 1 min the split valve was opened (75 mL min−1) and the injector temperature was kept at 260 °C. The injection volume was 1 μL. The mass spectra detector (MSD) operated in selected ion monitoring (SIM) mode. Retention times and ions monitored for each compound are summarized in Table 1. The electron multiplier was set at a nominal value of 1459 V. Linearity was assessed by preparing standard solutions in chloroform from 0.1 to 1000 ng mL−1. The GC–MS/MS analysis was performed using a Thermo Scientific Trace 1310 gas chromatograph coupled to a triple quadrupole mass spectrometer (TSQ 8000) and an IL 1310 autosampler from Thermo Scientific (San Jose, CA, USA). Separation was carried out on a 5% phenyl-arylene/95%dimethylpolysiloxane Zebron ZB-SemiVolatiles capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) supplied by Phenomenex (Torrance, CA, USA). Helium (purity 99.999%) was employed as carrier gas at a constant column flow of 1.0 mL min−1. The GC oven temperature was programmed from 100 °C (held 1 min) to 290 °C at 25 °C min−1 (held 4 min). The injector temperature was 260 °C. Injector was operating in the splitless mode and programmed to return to the split mode after 1 min from the beginning of a run. The mass spectrometer (MSD) was operated in the electron impact (EI) ionization positive mode (+70 eV). The temperatures of the transfer line and the ion source were set at 290 and 350 °C, respectively.

Selected reaction monitoring acquisition mode (SRM) was implemented. Quantification was performed using the Trace Finder software. Linearity was assessed by preparing standard solutions in chloroform starting from the solution from 0.1 to 1000 ng mL−1. 2.4. Statistical analysis Basic and descriptive statistics, as well as experimental design analysis, were performed using Statgraphics Centurion XVI 16.1.15 (Manugistics, Rockville, MD, USA) as software package. Experimental design methodology was applied for the optimization of the USAEME process to evaluate the simultaneous effects of the experimental parameters. 3. Results and discussion 3.1. Chromatographic analysis Individual standards of each target compound and the internal standard (PCB-30) in ethyl acetate were analyzed by GC–MS in the full scan mode to find out the retention times and the best ions to perform the single ion monitoring (SIM) detection. Three ions were selected for each compound: one for quantification purposes and two for confirmation. The retention times and the ions monitored for each compound are summarized in Table 1. MS/MS detection offers some advantages such as the matrix interference removal, provides reliable confirmation and allows the selective quantitation of target compounds in high background samples. All these benefits are due to that MS/MS transitions are characteristic for each compound, and they are hardly coinciding with the ones of the others substances. MS/MS working conditions were optimized using the automated selected reaction monitoring (AutoSRM) tool implemented in the TSQ8000 GC–MS/MS software. In this automated process, three different steps are involved: (i) pre-cursor ion study; (ii) product ion study; and (iii) SRM optimization. Thus, working in electron ionization (EI) mode, the precursor ion was identified by full scan mode for each compound. In a second step, the mass spectrometer was set to product-ion-scan mode and the most intense precursor ions were selected for subsequent fragmentation with different collision energies

Table 1 Experimental GC–MS and GC–MS/MS parameters for the analysis of the target UV filters. Acronym

INCIa name

CAS number

GC–MS analysis RT (min)

Selected ions (m/z)

GC–MS/MS analysis c

RT (min)

Selected quantification transition (m/z)

Selected confirmation transitions (m/z) 186.0 N 150.4 (25) 256.0 N 185.9 (25) 120 N 92 (10) 250.1 N 120 (15) 91 N 65 (15) 120 N 92 (10) 262.2 N 120 (15) 161 N 133 (10) 248.1 N 178 (10) 127.9 N 102 (20) 170.6 N 128.1 (15) 119 N 91.8 (10) 275.2 N 137.0 (10) 231.9 N 176.5 (20) 248 N 164.9 (25) 148 N 104.2 (25) 165.1 N 148.6 (25) 161 N 133.1 (10) 290.2 N 178.1 (10) 232 N 203 (20) 360.2 N 276.1 (20)

PCB-30

2,4,6-Trichlorobiphenylb

35693-92-6

6.95

150.0, 186.0, 256.0

6.89

257.9 N 185.9 (20)

EHS

Ethylhexyl salicylate

118-60-5

7.08

120.0, 138.0, 250.1

6.85

138 N 120 (10)

BS HMS

Benzyl salicylate Homosalate

118-58-1 118-56-9

7.41 7.47

65.0, 91.1, 228.1 109.1, 124.1, 138.0

7.21 7.22

228.1 N 91 (10) 138 N 120 (10)

IAMC

Isoamyl methoxycinnamate

71617-10-2

8.02

161.1, 178.1, 248.0

7.83

178.1 N 161.1 (10)

4MBC

4-Methylbencylidene camphor

36861-47-9

8.18

211.1, 239.1, 254.0

7.97

254.1 N 239.2 (10)

MA

Menthyl anthranilate

134-09-8

8.38

92.0, 119.0, 275.2

8.16

137 N 119 (10)

Eto

Etocrylene

5232-99-5

8.59

232.1, 248.1, 277.1

8.31

276.9 N 248.1 (10)

EHPABA

Ethylhexyl dimethyl PABA

21245-02-3

8.88

148.0, 165.0, 277.2

8.65

277.2 N 164.9 (10)

2EHMC

2-Ethylhexyl methoxycinnamate

5466-77-3

9.03

161.0, 178.0, 290.0

8.80

177.9 N 133.1 (20)

OCR

Octocrylene

6197-30-4

10.70

232.0, 249.0, 360.0

10.17

a b c

International Nomenclature of Cosmetic Ingredients. IUPAC name. Values underlined are ions that were used for quantification purposes.

248 N 165 (30)

M. Vila et al. / Microchemical Journal 124 (2016) 530–539

150000

533

MA

EHS 100000

100000

137

119

120

138

HMS

50000

50000 0

0

40000

50000

Eto 228.1

248.1

BS

20000

Abundance

0

0

Abundance

276.9

91

40000

178.1

IAMC

100000

EHPABA

50000

277.2

164.9

177.9

133.1

0

161.1

150000 20000

2EHMC

100000 0

50000

0 40000

4MBC

200000

254.1

OCR

239.1

248

20000

100000 0

165

0

6.5

7.0

7.5

8.0

8.5

8.0

8.5

9.0

9.5

10.5 Time (min)

10.0

Fig. 1. Reconstructed SRM chromatogram of a real sample (river water) spiked with the target compounds (0.1 μg L−1).

two levels: extraction temperature at 25 and 50 °C, time at 5 and 15 min, and the addition of salt at 0 and 20% (w/v). To carry out the design, 10 mL of Milli-Q water was placed in 15 mL conical-bottom glass centrifuge tubes and spiked with 100 μL of a solution of 100 ng mL−1 in acetone of the target analytes to reach a final sample concentration of 1 ng mL−1. Prior to the addition, a pair of spatulas of sodium thiosulfate (~100 mg) was added. Then, if needed, the salt was included. Subsequently, 100 μL of the corresponding extracting solvent containing 10 ng of PCB-30 (used as internal standard to correct the differences of the extraction solvent volume) was incorporated. Finally, the USAEME process was implemented as reported in Section 2.2, but modifying the extraction temperature and time according to the experience. The results for the multifactor ANOVA study are shown in Table 2. Taking into account the high F-ratios and low p-values, the solvent (A) was the most influential variable, resulting statistically significant for the majority of the analytes. The temperature (B) and the time (C) were significant in four cases, while the addition of salt was significant for three compounds. The interactions solvent-temperature (AB),

in the collision cell (Q2). Finally, the optimization of the collision energy for each transition was conducted. Two or three transitions were selected for each target compound and for the internal standard (PCB-30) according to ion abundance. The most intense transition was used for quantification purpose, whereas the second and the third ones were employed for identification/confirmation purposes (Table 1). Fig. 1 shows a SRM chromatogram of a real sample (river water) spiked with the target compounds (0.1 μg L−1). 3.2. Optimization of the extraction process The influence of the main variables potentially affecting the USAEME must be evaluated to achieve an efficient extraction. In this way, the extracting solvent (A), the extraction temperature (B), the extraction time (C), and the addition of salt (NaCl) (D) were studied by means of a multifactor experimental design 3 × 23. Three different solvents were examined: trichloroethane (TCE), carbon tetrachloride (CCl4) and chloroform (CLF). The rest of the parameters were explored at

Table 2 ANOVA summary table obtained for the USAEME process. Compound

EHS BS HMS IAMC 4MBC MA Eto EHPABA 2EHMC OCR

Solvent (A)

Temperature (°C) (B)

Time (min) (C)

NaCl (%) (D)

AB

AC

AD

F

p

F

p

F

p

F

p

F

p

F

p

F

p

65.41 23.04 46.40 22.24 71.07 6.54 98.45 3.27 3.57 0.71

0.000 0.000 0.000 0.000 0.000 0.018 0.000 0.086 0.072 0.517

1.60 0.06 6.03 6.57 33.96 3.64 17.56 2.47 3.02 3.65

0.237 0.816 0.036 0.031 0.000 0.089 0.002 0.150 0.116 0.088

0.14 4.75 3.55 0.31 0.01 4.85 11.2 6.08 7.45 6.23

0.720 0.057 0.092 0.591 0.907 0.055 0.009 0.036 0.023 0.034

2.02 5.63 0.72 0.69 5.81 0.42 39.62 0.58 0.34 1.92

0.189 0.042 0.419 0.429 0.040 0.532 0.000 0.466 0.574 0.199

3.23 1.45 2.19 2.34 4.41 0.23 8.73 0.88 10.5 5.22

0.088 0.285 0.168 0.152 0.046 0.803 0.008 0.449 0.004 0.031

9.31 2.24 6.02 2.66 2.33 0.13 1.51 0.37 4.24 0.92

0.006 0.163 0.022 0.124 0.153 0.880 0.272 0.702 0.051 0.434

1.85 4.21 4.19 5.15 25.4 0.01 34.2 1.05 0.31 1.90

0.213 0.051 0.052 0.032 0.000 0.994 0.000 0.391 0.739 0.205

Values in bold show significative factors.

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M. Vila et al. / Microchemical Journal 124 (2016) 530–539

solvent-time (AC), and solvent-salt (AD) were significant for four, two and three compounds, respectively. The interactions temperaturetime (BC), temperature-salt (BD) and time-salt (CD) were never significant. The results are graphically depicted in the means plot charts shown in Fig. 2a for some representative analytes. As regards the extracting solvent, it can be seen that chloroform offers the highest responses for most of the compounds, while TCE provides the best results only for three compounds, and carbon tetrachloride gave the lowest responses. A time of 5 min affords higher results for six compounds and a time of 15 min, for the other five compounds. Taking into account that only in four cases this factor was statistically significant and in three of these cases the best results were obtained with 5 min of extraction,

a)

2EHMC

4MBC (X 1000)

(X 1000)

77 73 69 65 61 57

104 94 84 TCE

CCl4 Solvent

74

CLF

TCE

10.5 10 9.5 9 8.5 8

25 50 Temperature

(X 1000)

(X 1000)

68

107

67

97

66

87

65

5

Time

5

(X 1000) 99 96 93 90 87 84

69 67 65 63

0

NaCl

0

20

Time

NaCl

15

20

2EHMC

4MBC Temperature 25 50

(X 1000)

Temperature

(X 1000)

25 50

108 98

88 78 TCE

CCl4 Solvent

68

CLF

TCE

CCl4 Solvent

CLF

Eto

HMS (X 1000) 17

Time 5

(X 1000)

NaCl

39

15

36

0 20

15

33

13

30

11

CLF

25 50 Temperature

77

15

(X 1000)

78 73 68 63 58 53

CCl4 Solvent

(X 10000)

(X 1000) 77 73 69 65 61 57

b)

the lowest time value was chosen. The addition of salt gave higher responses although it was only significant for three analytes (e.g. 2EHMC). Therefore, salt (20% NaCl, w/v) was selected to perform the extractions. After analyzing the mean plots for the temperature, it can be noted that extractions at 50 °C gave greater outcomes. However, looking at the interaction plots other conclusions can be drawn (Fig. 2b). The pair of lines for each interaction corresponds to the predicted response when a factor varies from its low to high level, in each level of the other factor and keeping the remaining factors (not involved in the interaction) in its central level. The interaction solvent-temperature (AB) was important in four cases (4MBC, Eto, 2EHMC and OCR). It can be observed (see Fig. 2b) for 4MBC and 2EHMC) that, employing chloroform

27 TCE

CCl4 Solvent

CLF

TCE

CCl4 Solvent

CLF

Fig. 2. a) Mean plots for extracting solvent, temperature, time and salt for some representative UV filters and b) interaction plots for solvent-temperature (AB), solvent-time (AC) and solvent-salt (AD) for some representative compounds.

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Table 3 Linearity, limits of detection and quantification and precision achieved with the proposed method. Compounds

LOD (ng mL−1)

Linearity

LOQ (ng mL−1)

Range (ng mL−1)

R2

IDL (ng mL−1)

a.) GC–MS EHS BS HMS IAMC 4MBC MA Eto EHPABA 2EHMC OCR

1–1000 10–1000 5–1000 2.5–1000 0.1–1000 2.5–1000 2.5–1000 2.5–1000 1–1000 10–1000

0.9988 0.9974 0.9994 0.9971 0.9976 0.9995 0.9992 0.9989 0.9983 0.9988

0.20 2.7 1.5 0.58 0.022 0.58 0.38 0.54 0.21 2.5

0.0030 0.027 0.015 0.0058 0.00022 0.0058 0.0038 0.0054 0.0021 0.025

0.010 0.090 0.050 0.019 0.00073 0.019 0.013 0.018 0.0070 0.083

b.) GC–MS/MS EHS BS HMS IAMC 4MBC MA Eto EHPABA 2EHMC OCR

0.1–1000 0.5–1000 0.5–1000 0.5–1000 0.1–1000 0.5–1000 0.5–1000 0.1–1000 0.5–1000 0.5–1000

0.9923 0.9910 0.9925 0.9956 0.9994 0.9979 0.9980 0.9978 0.9977 0.9961

0.029 0.10 0.090 0.10 0.029 0.15 0.040 0.0080 0.060 0.050

0.00029 0.0010 0.0013 0.0010 0.00029 0.0015 0.00040 0.000080 0.00066 0.00050

0.00097 0.0033 0.0043 0.0033 0.0097 0.0050 0.0013 0.00027 0.0022 0.0017

Precision RSD % 10 ng mL−1

as extracting solvent, there was no difference between the two temperatures. Operating at 25 °C (e.g. 2EHMC) was even more favorable. The same occurred for the interaction solvent-time (AC), which was statistically significant for two compounds (EHS and HMS). The interaction plots for these compounds (see the graph for HMS in Fig. 2b) show that while using TCE or CCl4 the difference between both times is important, utilizing chloroform the difference is minimum. Therefore, the shortest time was chosen. The other interaction that emerged significant was the interaction solvent-salt (AD), especially for three compounds (IAMC, 4MBC and Eto). Again, as can be seen in Fig. 2b for Eto, there is not a significant difference between adding salt or not when the extraction is performed with chloroform, while for the other two solvents there is a substantial difference. Finally, an addition of 20% of salt was selected because, in this way, the volume of the obtained extract was largest than without salt, allowing a better manipulation of it. The rest of the interactions were not significant. In brief, the operating conditions proposed to perform the USAEME were based on the use of chloroform as extracting solvent, at 25 °C, during 5 min and with 20% (w/v) of salt. 3.3. Method performance Regarding the instrumental linearity, calibration standards were prepared in chloroform covering a concentration range from 0.1 to 1000 ng mL−1 (see specific ranges in Table 3 for GC–MS andGC–MS/

100 ng mL−1

750 ng mL−1

2.3 4.8 3.6 6.3 4.5 6.9 3.0 8.4 6.1 6.4

1.8 4.8 3.4 5.0 4.5 3.5 4.3 5.7 7.5 5.4

5.9 9.3 5.7 7.8 3.4 4.9 3.8 5.4 5.0 8.0

3.1 10.2 7.2 7.2 5.6 6.4 1.8 3.6 5.5 6.2

3.0 0.8 2.2 3.1 1.9 1.7 1.4 1.1 1.6 0.8

1.4 1.4 1.3 0.3 0.2 0.9 1.3 1.6 2.5 7.0

MS analysis) with thirteen levels and three replicates at each level. Both methods exhibited a direct proportional relationship between the amount of each analyte and the chromatographic response. The linearity was equivalent with both detectors, obtaining determination coefficients R2 ≥ 0.9971 for GC–MS and R2 ≥ 0.9910 for GC–MS/MS in all cases. The instrumental detection limits (IDLs) were calculated as the concentration giving a signal-to-noise of three (S/N = 3). IDLs forGC–MS were lower than 0.6 ng mL−1 for the majority of the compounds (excluding HMS, 1.5 ng mL− 1; BS, 2.7 ng mL− 1 and OCR, 2.5 ng mL−1), while for GC–MS/MS they were in all cases lower than 0.15 ng mL−1, decreasing, therefore, at least one order of magnitude for the majority of the compounds utilizing tandem mass spectrometry. Precisions for GC–MS and GC–MS/MS were evaluated with standards at three different concentrations (10, 100 and 750 ng mL−1), analyzing each one between three and ten times. The intraday precision was between 1.8 and 9.3% for GC–MS, while for GC–MS/MS it was between 0.2 and 7.2% (except in one case). The interday precision was between 2.3 and 11% for GC–MS and between 3.4 and 11.0% for GC–MS/MS. Therefore, precision was similar for both GC–MS and GC–MS/MS analysis. To assess the performance of both methods (USAEME–GC–MS and USAEME–GC–MS/MS), analytical quality parameters were evaluated. The limits of detection and quantification (LODs and LOQs) were calculated as the compound concentration giving a signal-to-noise ratio of three (S/N = 3) and ten (S/N = 10), respectively, in a real sample and

Table 4 Recoveries (SD) (%). Recovery (RSD)% n = 4

EHS BS HMS IAMC 4MBC MA Eto EHPABA 2EHMC OCR

0.025 ng mL−1

0.1 ng mL−1

1 ng mL−1

River water

River water

Tap water

Sea water

Swimming pool water

Mean recovery

Milli-Q water

Tap water

Sea water

SPA water

Mean recovery

93.1 (12.9) 94.1 (4.4) 96.8 (11.4) 88.9 (2.5) 69.6 (13.7) 28.7 (14.9) 84.6 (12.1) 64.7 (12.7) 98.5 (7.5) 102 (12.8)

105 (5.4) 101 (13) 98.4 (2.2) 94.4 (9.9) 95.9 (5.3) 102 (5.4) 100 (8.9) 93.5 (0.88) 99.9 (8.4) 105 (7.7)

110 (1.8) 94.0 (4.3) 92.3 (1.2) 106 (10) 98.5 (4.2) 68.2 (2.5) 105 (3.7) 72.9 (6.0) 96.2 (8.6) 97.4 (14)

108 (4.8) 108 (6.3) 91.5 (3.3) 108 (3.5) 96.7 (3.8) 97.7 (1.8) 108 (6.2) 84.2 (11) 102 (6.0) 102 (4.1)

97.9 (2.3) 96.9 (4.9) 90.8 (3.4) 95.1(3.8) 97.1 (3.6) 81.0 (12) 106 (4.8) 83.0 (15) 91.0 (8.3) 101 (1.9)

105 (3.6) 99.9 (7.1) 93.2 (2.5) 101 (6.8) 97.0 (4.2) 87.2 (5.4) 105 (5.9) 83.4 (8.2) 97.3 (7.8) 102 (6.9)

89.9 (7.7) 94.1 (7.1) 87.3 (5.6) 94.2 (5.2) 88.1 (1.9) 68.9 (10) 95.7 (2.3) 62.4 (2.4) 80.5 (7.3) 88.3 (9.3)

90.3 (3.5) 94.3 (5.6) 89.0 (5.0) 99.7 (3.9) 94.0 (10) 70.1 (7.8) 100 (2.9) 61.7 (13) 79.6 (4.8) 86.4 (4.3)

98.0 (5.5) 98.6 (6.9) 94.2 (12) 98.4 (6.3) 92.4 (9.7) 79.1 (10) 98.6 (4.8) 75.5 (8.1) 90.8 (9.9) 97.7 (3.3)

90.9 (7.4) 104 (2.7) 88.4 (7.9) 99.1 (2.9) 89.8 (11) 73.5 (7.3) 99.6 (4.4) 59.5 (13) 80.3 (4.8) 98.3 (12)

92.3 (6.0) 97.7 (5.6) 89.7 (7.6) 97.9 (4.6) 91.1 (8.2) 72.9 (8.8) 98.5 (3.6) 64.8 (9.1) 82.8 (6.7) 92.7 (7.2)

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Table 5 Comparison of the proposed USAEME–GC–MS and USAEME–GC–MS/MS method with other methods for the determination of UV filters in water samples. Method

Analytes

Linearity (μg L−1)

LOQ (ng L−1)

Extraction time (min)

Precision (RSD, %)

Recovery (%)

Reference

PDT, PBSA, BP4, BP3, 4MBC, IAMC, OCR, EHPABA, BMDBM EHS, HMS, IAMC, 4MBC, BP3, EHMC, EHPABA, OCR, BMDBM EHS, HMS, BS, IAMC, BP3, 4MBC, EHPABA, EHMC, OCR

5–2500 10–4000 10–10000

0.6–212 0.5–10 2–14

6–16 b13 9–14

63–122 89–115 80–117

[13] [14] [19]

BH, BP, EHS, HMS, BP3, BP1 EHS, BS, HMS, IAMC, 4MBC, MA, Eto, EHPABA, 2EHMC, OCR

0.05–10 10–1000 0.5–1000

20–100 0.73–100 0.27–4.8

180 30 1 + 3 (shaken + centrifugation) 4 5 + 10 (extraction + centrifugation)

6.1–12.9 2.3–11 3.4–11

76.5–120 69.6–110

[21] This study

Number

Acronym

SBSE–TD–GC–MS SPME–GC–MS/MS DLLME–GC–MS

9 9 9

VADLLME–GC–MS USAEME–GC–MS USAEME–GC–MS/MS

6 10

PDT: phenyldibenzimidazoletetrasulfonic acid; PBSA: 2-phenylbenzimidazole-5-sulfonic acid; BP4: benzophenone-4; BP3: benzophenone-3; BMDBM: butyl-methozydibenzoylmethane; BH: benzhydrol; BP: benzophenone

taking into account the concentration factor. LODs were in the range of parts per trillion, which is necessary for the determination of UV filters in water samples since they are present at trace levels. LODs were lower than 0.027 ng mL−1, while LOQs were lower than 0.090 ng mL−1 for all the analytes. However, as expected, the use of MS/MS allowed getting lower LODs and LOQs than employing a simple quadrupole. LODs were lower than 0.0015 ng L− 1, whereas LOQs were lower than 0.0050 ng L−1 for all the target compounds. This means a difference of an order of magnitude between the two mass spectrometry techniques. Diverse water samples such as Milli-Q water, tap water, seawater, spa water, swimming-pool water and river water were employed to test the suitability of the method in different matrices. These samples were fortified with known concentrations of each target compound at three levels: 0.025 ng mL−1, 0.1 ng mL−1 and 1 ng mL−1. USAEME procedure was implemented as indicated in Section 2.2, but adding 100 μL of a solution containing the target compounds in acetone at 2.5 ng mL−1 (0.025 ng mL−1 level), 10 ng mL−1 (0.1 ng mL−1 level) or 100 ng mL−1 (1 ng mL−1 level) before the chloroform addition. Recovery values were

Table 6 Concentration (ng mL−1) of the target UV filters in the analyzed water samples. GC–MS

EHS

SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 SP10 SP11 SP12 AP1 AP2 AP3 AP4 SW1 SW2

0.10 0.14 0.14 0.10 0.10 0.14 0.24 0.15 141 0.16 0.090 2.5 22 0.24

BS

HMS

IAMC

4MBC

Eto

0.0013 0.0027 0.0012 0.0014 0.0018 0.0086 0.14 1.2

0.19

0.16 0.23 0.21

0.42

2.6 27

0.093 3.7

0.72

0.0027 0.0012 0.0050 0.059

0.041

0.088

GC–MS/MS

EHS

BS

HMS

SP13 SP14 SP15 AP5 SPA1 TW1 RW1 RW2 RW3 RW4 RW5

0.015 0.093 0.053 0.44

0.0072 0.13 0.020 0.023

0.012 0.031 0.085 0.68

0.027 3.3 0.0092 0.016 0.086 89

0.029 0.015 0.025 0.010 0.059

0.033 0.0080 0.0071 0.010 0.035 0.072

IAMC

OCR

0.016 0.021 0.036 0.012 0.023 0.028 0.034 0.025 0.011 0.19 0.16 0.078 0.46 1.9 0.019 0.030

0.42 0.13 0.94

1.2 4MBC

0.0036 0.0037 0.0034

3804 0.14 0.13 169 155 0.20 0.20 1.1 171

2EHMC

OCR

0.010

0.0076 0.039 0.016 0.10 0.043 0.0087 0.0080

0.24 0.15 0.75 31 0.21 0.030 3.8 0.035 0.049 0.27 323

0.036 0.0068

0.13 0.12 0.17 0.31

Eto

0.0028 0.015 0.029 0.080 0.13

2EHMC

0.024

0.012

SP: swimming pool water; AP: aquapark SW: seawater (bathing area); SPA: spa water; TW: tap water; RW: river water (RW5 bathing area); blank spaces: below LODs.

calculated as the ratio of the response obtained for spiked samples to the response measured for standards (at the same concentration level) and expressed as percentage (Table 4). Good recoveries and satisfactory precision were achieved. Recoveries were higher than 90% in most cases excluding few exceptions (MA and EHPABA) in some samples and precisions were between 0.9 and 15%. The lowest level (0.025 ng mL− 1) was only analyzed by GC–MS/MS and recoveries were also good except for MA. Consequently, USAEME can be considered as an exhaustive microextraction technique. Accordingly, concerning the recoveries, both techniques gave similar results. Regarding matrix effects, equivalent responses were obtained for the different types of water. Therefore, it can be assumed that no matrix effects exist and quantification can be performed by external calibration using standards prepared in chloroform. This is a great advantage of the proposed methods compared with other works in which all the extraction process needs to be performed to obtain the calibration curve [19]. 3.4. Comparison of USAEME with other sample preparation techniques Differences between diverse kinds of sample preparation such as SPE [8], SBSE-TD [13], SPME [14], DLLME [19] and VADLLME [21] can be observed regarding the analytical parameters, the extraction time or the sample volume utilized. In Table 5 the analytical performance (linearity, LOQs, and %RSDs) of the present method is compared with other methods described in the literature with the same finality, and, as can be seen, the values reported are at the same level or range. LOQs were at the ng L−1 level, with values between 0.27 and 212 ng L−1, resulting SPME–GC–MS/MS, DLLME–GC–MS and USAEME–GC–MS/MS methods those with the lowest LOQs. Regarding recoveries, all the compared studies show values between 66 and 122%. In the present study, recoveries between 69 and 110 were achieved; thus these six methods are comparable. However, the vast part of the methods require a standard addition calibration [20] or, at least, to prepare a calibrate carrying out the hole process to perform the quantitation [13–15,19], complicating and making tedious the quantitation, whereas, in the case of USAEME, the calibration with solvent standards can be easily applied. The USAEME procedure is a simple technique that allows to obtain an efficient extraction in a short time. Only two principal steps are needed (ultrasound emulsification and centrifugation), making it an easy sample preparation. No additional steps such as concentration are needed, as can be necessary with SPE [8]. In comparison with SPE, SBSE and SPME, the extraction time for USAEME is greatly shortened from times N30 min (3 h for SBSE [13]) to 5 min for USAEME since the extraction can reach equilibrium very rapidly due to the power of ultrasounds. Regarding the sample volume, it needed lesser amount of sample for SPE process (e.g. 200 mL [8]). Compared with DLLME, no disperser solvent was required in USAEME. No significant differences between VADLLME and USAEME are appreciated. Another advantage of USAEME is the low cost of the extraction, due to only a few microliters of the extracting solvent is needed;thus,it practically avoids the use of solvent that is necessary in other techniques

M. Vila et al. / Microchemical Journal 124 (2016) 530–539

537

twenty-nine different water samples from various origins and with different matrix composition (swimming pools, aquaparks, seawater, spas, rivers…) were analyzed (Table 6). It should be pointed out that, in some cases, the dilution of the samples with Milli-Q water was performed to get the corresponding extracts with concentrations within the calibration range. The target UV filters most often found were OCR, 2EHMC and EHS, with twenty-seven (93%), twenty-six (90%) and twenty-five (86%) positive samples, respectively. The UV filter that turned up in the highest concentrations was OCR, followed by EHS, and, then, by HMS. All these compounds are commonly included in sun protection products. In addition the largest concentrations were found in open-air swimming pools, aquaparks and seawater in summer (see AP1, AP2, SP10 and SW2 in Table 6), where people apply too much sunscreens to protect themselves from solar rays. In these places, the water remains

(LLE, SPE). Furthermore, the technique does not require special devices such as SPE cartridges, SPME fibers or coated stir bars. Some disadvantages of the proposed method are the use of chloride solvent, although it is used in small quantities (0.1 mL), and the difficulty for automatizing, comparing with SPME or SPE, mainly due to the centrifugation step. In very few cases, analyte degradation caused by the influence of ultrasonic energy may occur [27]. In brief, USAEME is a fast, environmentally friendly, highly efficient, reproducible, simple and cheap microextraction technique for the enrichment and determination of UV filters in water samples. 3.5. Application to real samples In order to evaluate the suitability of the developed USAEME–GC– MS method for the analysis of UV filters in real water samples,

a) OCR

240000 220000 200000 180000

Abundance

160000 140000

HMS 120000

EHS

100000 80000 60000 40000

IAMC 4MBC

20000

2EHMC

0 7.00

7.50

8.00

8.50

9.00

9.50

10.00

10.50

Time (min)

b) 20000

EHS

800000

138

120

15000

HMS

4MBC

254.1

239.1

276.9

248.1

177.9

133.1

10000

400000

5000 0

0

Abundance

Abundance

12000

15000

BS 228.1

10000

Eto

8000 4000

91

0 5000

200000

0

2EHMC 100000

0 12000000

IAMC 178.100

161.1

200000

8000000

100000

4000000

248

165

OCR

0

0 6.5

7.0

7.5

8.0

8.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0 Time (min)

Fig. 3. a) Total ion chromatogram (GC–MS) and b) reconstructed SRM chromatogram (GC–MS/MS) for two aquapark waters (AP2 (a) and AP5 (b)) containing both eight targets (see concentrations in Table 6).

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M. Vila et al. / Microchemical Journal 124 (2016) 530–539

stagnant and, consequently, UV filters can accumulate and reach high concentrations. Sometimes, a large number of UV filters simultaneously appeared in a unique sample. It was the case for swimming pool and aquapark waters in which between 5 and 8 compounds were detected. MA and EHPABA did not appear in any water sample, but it is worth mentioning that the European Regulation forbids the first one in cosmetics [1] and the use of the second one is not extended. A remarkable fact is that etocrylene was found at levels of parts per trillion in two samples in spite of being prohibited in Europe in cosmetic samples [1]. It stands out, hence, the high occurrence frequency of the UV filters in water samples as well as the high concentrations they can reach, above all in swimming pools and aquaparks. A SIM reconstructed chromatogram of an aquapark water (AP2) is shown in Fig. 3a. In this sample, eight out the ten compounds were found (concentrations are given in Table 6). A 1:20 dilution of the sample was needed to quantify some of the targeted compounds that exceeded the maximum concentration included in the calibration range. The SRM reconstructed chromatogram of a different aquapark sample (AP5) is displayed in Fig. 3b also showing the presence of eight UV filters.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

4. Conclusions A methodology based on ultrasound-assisted emulsification– microextraction (USAEME) followed by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed for the simultaneous analysis of different classes of UV filters, including methoxycinnamates, salicylates, p-aminobenzoic acid derivatives, and others in water samples. The extraction parameters were optimized by means of experimental design tools. The selected conditions for the USAEME process were 100 μL of chloroform, an extraction temperature of 25 °C, 20% (w/v), an extraction time of only 5 min and a sample amount of 10 mL. Good linearity (R2 N 0.9910), quantitative recoveries (N 90% in most cases) and satisfactory precision (RSD b 10% in most cases) were achieved under the optimal conditions. Better results were gained with tandem mass spectrometry regarding limits of detection, enabling to determine lower concentrations of the target compounds, as required for this kind of emerging contaminants present at trace levels in water. Finally, the validated methodologies were successfully applied to the analysis of different water samples coming from swimming pools, aquaparks, rivers, seawater and spas. Since no significant matrix effects could be observed, quantification could be easily performed by external calibration using standards in chloroform, allowing a high analytical throughput and a valuable protocol simplification. The most frequent compounds found were OCR, 2EHMC and EHS. OCR and the salicylates EHS and HMS came out with the highest concentrations, particularly in open-air swimming pools and aquaparks.

[11] [12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

[21]

Acknowledgements This research was supported by European Regional Development Fund (ERDF) (2007–2013), UNST10-1E-491 (Infrastructure Program, Ministry of Science and Innovation) and projects CTQ2013-46545-P (Ministry of Economy and Competitiveness, Spain) and GPC2014/035 (Consolidated Research Groups Program, Xunta de Galicia). M.V. would like to acknowledge the Gil Davila Foundation for her grant, Xunta de Galicia for a predoctoral fellowship and the Ministry of Education, Culture and Sport for a FPU grant.

[22]

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[24]

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