Water with Low Concentration of Surfactant in Dispersed Solvent-Assisted Emulsion Dispersive Liquid−Liquid Microextraction for theDetermination of Fungicides in WineWan-Chi Tseng,†,∥ Shang-Ping Chu,†,∥ Po-Hsin Kong,§ Chun-Kai Huang,# Jung-Hsuan Chen,⊥

Pai-Shan Chen,*,§,⊥ and Shang-Da Huang*,†

†Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan§Department and Graduate Institute of Forensic Medicine, National Taiwan University, Taipei 10002, Taiwan#Department of Chemistry, National Chung-Hsing University, Taichung 40227, Taiwan⊥Forensic and Clinical Toxicology Center, National Taiwan University College of Medicine and National Taiwan University Hospital,Taipei 10051, Taiwan

ABSTRACT: A sample preparation method, dispersive liquid−liquid microextraction assisted by an emulsion with lowconcentration of a surfactant in water and dispersed solvent coupled with gas chromatography−mass spectrometry, wasdeveloped for the analysis of the fungicides cyprodinil, procymidone, fludioxonil, flusilazole, benalaxyl, and tebuconazole in wine.A microsyringe was used to withdraw and discharge a mixture of extraction solvent and 240 μL of an aqueous solution of TritonX-100 (the dispersed agent) four times within 10 s to form a cloudy emulsion in the syringe. This emulsion was then injectedinto a 5 mL wine sample spiked with all of the above fungicides. The total extraction time was approximately 0.5 min. Underoptimum conditions using 1-octanol (12 μL) as extraction solvent, the linear range of the method in analysis of all six fungicideswas 0.05−100 μg L−1, and the limit of detection ranged from 0.013 to 0.155 μg L−1. The absolute recoveries (n = 3) and relativerecoveries (n = 3) were 30−83 and 81−108% for white wine at 0.5, 5, and 5 μg L−1, and 30−92 and 81−110% for red wine,respectively. The intraday (n = 7) and interday (n = 6) relative standard deviations ranged from 4.4 to 8.8% and from 4.3 to11.2% at 0.5 μg L−1, respectively. The method achieved high enrichment factors. It is an alternative sample preparation techniquewith good performance.

KEYWORDS: water with low concentration of surfactant, dispersive liquid−liquid microextraction, fungicides, gas chromatography,improved solvent collection system, wine

■ INTRODUCTIONDefending against fungal diseases is the main challenge duringgrape growing for winemaking. To improve the quality andquantity of grapes, a broad spectrum of fungicides is frequentlyapplied during grape cultivation. Although some of them mightbe human carcinogens investigated by the U.S. EnvironmentalProtection Agency,1 without the use of fungicides in viticulture,large economic losses may be incurred. Under proper use,fungicides have minimal adverse impact on the environment orpublic health.2,3 However, improper treatment, that is, disregardof reasonable doses and safety periods, leads to fungicideresidues in the food chain, soil, and water because of theirhigh mobility and water solubility. This makes them commonenvironmental pollutants.4 In grapes, these residues maybe transferred to the must and then to the wine duringfermentation, which can be a significant issue for publichealth.5−13 The maximum residue levels for fungicides in grapesare set at 0.05−0.1 mg kg−1 by the European Union.14 InTaiwan, the maximum residue concentrations of cyprodinil,procymidone, fludioxonil, flusilazole, benalaxyl, and tebucona-zole in grapes are 1, 2.0, 1, 0.5, 0.5, and 2.0 mg kg−1,respectively.15 The tolerance for procymidone in wine is set at5 mg L−1 by the U.S. Food and Drug Administration (FDA).16

Because fungicides in matrices usually occur in trace amounts,sample pretreatment for chromatography is essential to analysis.

To achieve low limits of detection (LODs), a variety oftechniques such as liquid−liquid extraction,17,18 membrane-assisted solvent extraction,19 solid-phase microextraction,20

solid-phase extraction,21−24 and bar adsorptive microextraction25

have been developed to determine fungicides in wine samples.However, the main disadvantage of these methods is that theyrequire considerable time for extracting target analytes into anorganic phase or onto sorbents. Therefore, a fast, sensitive, andefficient analytical method is needed.Recently, Rezaee et al. introduced dispersive liquid−liquid

microextraction (DLLME).26 This method injects a mixtureof a high-density extraction solvent and a water-miscible andpolar dispersive solvent into the aqueous sample to form acloudy mixture, which results in high enrichment. In 2007,Khalili-Zanjani et al. performed liquid−liquid microextractionbased on solidification of a floating organic droplet.27 Theextraction solvent, which had a melting point near roomtemperature (10−30 °C), was frozen by transferring the sampleto an ice bath after extraction. In 2011, Lee et al. introduced an

Received: April 2, 2014Revised: August 24, 2014Accepted: August 25, 2014Published: August 25, 2014

Article

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automated, dynamic in-syringe liquid-phase microextraction,which was more efficient for extracting pesticides from watersamples.28 Several studies have employed low-density solventsas substitutes for toxic halogenated solvents. These develop-ments widened the selection of solvents available for DLLMEand extended its application.17,29−34

Ultrasound-assisted emulsification microextraction (USAEME)for DLLME has also been developed to improve the generationof droplets with or without using dispersive solvent.34−38 Anotheralternative to ultrasound assistance is the use of surfactants. Assome surfactants are just soluble in an organic solvent and someare just water-soluble, their presence improves the generationof fine droplets in DLLME.39−41 However, high concentrationsof surfactants are generally required to disperse the extractionsolvents. Water with a low concentration of surfactant indispersed solvent-assisted emulsion dispersive liquid−liquidmicroextraction (WLSSAE-DLLME) was developed recently,42,43

which used in combination with an improved solvent collectionsystem (ISCS) avoids the problems associated with DLLME toanalyze water samples.33

In the present study, WLSSAE coupled with gas chromatog-raphy−mass spectrometry (GC-MS) was developed to analyzesix fungicides in wine samples. The accuracy, precision, linearity,enrichment factor (EF), and LOD of WLSSAE in the analysis ofwine samples were evaluated and tested.

■ MATERIALS AND METHODSReagents and Samples. All solvents and chemicals used in the

study were of analytical grade. Cyprodinil (CYP), procymidone(PRC), fludioxonil (FLD), flusilazole (FLU), benalaxyl (BEN),tebuconazole (TEB), anthracene-d10 (ANT-d10; internal standard),1-heptanol, 1-octanol, 1-nonanol, Triton X-114, Triton X-100, Tween80, and Tween 60 were purchased from Sigma-Aldrich (St. Louis,MO, USA). Methanol (liquid chromatography (LC)-MS grade) wasobtained from J. T. Baker (Phillipsburg, NJ, USA). Acetone (LCgrade) and sodium chloride were purchased from Merck (Darmstadt,Germany). Deionized water (DI water) was obtained by using a Milli-Qreagent water system (Millipore, Milford, MA, USA).Stock solutions of each fungicide and of ANT-d10 were prepared by

dissolving each in methanol to obtain a 1000 mg L−1 solution, whichwas then stored at 4 °C. Standard working solutions and ANT-d10solution were prepared by diluting each stock solution with methanolto 10 mg L−1. Each sample solution was prepared by spiking purewater with 10 and 5 μg L−1 fungicide and ANT-d10. Two kinds ofwhite wine and one kind of red wine made in California (USA) werepurchased from a local supermarket (Hsinchu, Taiwan). All winesamples contained 12% alcohol. The samples were filtered through0.45 μm membrane filters from Millipore (Bedford, MA, USA) andthen stored at 4 °C overnight before analysis.Instrumentation. To separate the aqueous and organic phases,

microtubes that were designed in-house (15 × 3 mm; inner diameter,1.8 mm; total volume, 38 μL; Qing-Fa Co., Hsinchu, Taiwan) wereused in the ISCS system. A CN-2200 centrifuge (Hsiantai MachineryIndustry, Taipei, Taiwan) and a miVac DUC-12060-C00 centrifuge(Stockholm, Sweden) were used in the study.Analyses were carried out by using a 6850 Agilent Technologies gas

chromatograph (Wilmington, DE, USA) with a split/splitless injectoroperated at 300 °C and a single quadrupole (Agilent Technologiesmass detector 5975B). The splitless time was 1 min. The flow rate ofhelium was 1.0 mL min−1. A 30 m DB-5MS UI-fused silica capillarycolumn (0.25 mm i.d., 0.25 μm film thickness; J&W Scientific, Folsom,CA, USA) was employed in the determination of fungicides. Thetemperature of the column was initially held at 100 °C, raised to 250 °Cat 35 °C min−1, held at 250 °C for 3 min, raised to 300 °C at 10 °Cmin−1, and then held at 300 °C for 1 min. The carrier gas washelium (purity = 99.9995%), which was further purified by passagethrough a helium gas purifier (Agilent Technologies model RMSH-2).

The temperature of the GC-MS transfer line was 280 °C, 230 °C forion source, and 150 °C for quadrupoles. A mass detector in the electronimpact mode (70 eV) was used. Spectra were scanned over the m/zrange of 50−400 to confirm the retention times of the analytes.Selected ion monitoring mode (SIM) was applied for the determinationof fungicides. In the determination of fungicides, two or three ions wereselected in Table 1.

Extraction Procedure. A diagram of the WLSSAE procedure isshown in our previously published work.42 A 5 mL portion of theblank white wine and DI water (1:1, v/v) spiked with analytes at5 μg L−1 were transferred to a 10 mL conical-bottom glass centrifugetube. A mixture of 12 μL of extraction solvent (1-octanol) and 240 μLof water containing 10 mg L−1 Triton X-100 (dispersed solvent) wastransferred to an Eppendorf tube. A 500 μL syringe (Reno, NV, USA)was used to pump the mixture back and forth four times within 10 s,and then a cloudy emulsion was formed. This emulsion was theninjected into the sample solution. After centrifugation for 5 min at5000 rpm, an organic droplet formed on the surface of the solution.The phase separation after DLLME of white and red wines is alsoshown in ref 44. The floating phase, which had a volume of approxi-mately 3.0 ± 0.5 μL, was hard to withdraw into a microsyringe.Therefore, the mixture of the floating phase and aqueous solution wasfirst transferred to a in-house-designed microtube (15 × 3 mm). Aftercentrifugation for 1 min, the organic phase was collected from theupper portion of the microtube by using a 10 μL microsyringe and theninjected into the GC-MS system. The ambient temperature was 25 °C.

■ RESULTS AND DISCUSSIONImpact of Solvent on Extraction Efficiency. The

selection of extraction solvent is important in maximizing theextraction efficiency. The solvents should be immiscible with

Table 1. Retention Time and m/z Values Selected for SIMMass Detection

compd retention time (min) selected ions (m/z)

ANT-d10 4.96 187, 188, and 189CYP 5.87 210, 214, and 225PRC 6.07 96, 283, and 285FLD 6.61 182 and 248FLU 6.76 206, 233, and 234BEN 7.78 91, 148, and 206TEB 8.22 120, 250, and 252

Figure 1. Effect of the type of extraction solvent (n = 3). Samples werespiked with 5 μg L−1 of each analyte. Extraction conditions: dispersedsolvent volume, 240 μL; surfactant, 1 mg L−1 Triton X-100.

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water and have low toxicity, and analytes should be highlysoluble in such solvents. To apply the above conditions,alcohols 1-heptanol, 1-octanol, and 1-nonanol were tested. Toconsistently collect 3.0 μL of the floating organic phase, variousamounts of extraction solvent (21 μL of 1-heptanol, 12 μL of1-octanol, and 11 μL of 1-nonanol) had to be combined with240 μL of Triton X-100 (1 mg L−1) in a microsyringe.43 Themixture was subsequently injected into the sample solution.The results are shown in Figure 1. The experiment revealedthat 1-octanol had better EFs than those of the other solvents;it was thus chosen as the extraction solvent for use insubsequent experiments.

Type of Surfactant. The selection of surfactant is crucial tothe success of the proposed method. As surfactants are solublein the extraction solvent and in water, they are commonly usedto improve the dispersion of extraction solvents. A surfactantwith high hydrophilic−lipophilic balance (HLB) has higherhydrophilicity. When the HLB value of a surfactant is between12 and 16, the surfactant is considered as an oil-in-wateremulsifier. Two kinds of well-known polyoxyethylene-typenonionic surfactants, Tween and Triton (Figure 2), werestudied. The HLBs of Tween 60, Tween 80, Triton X-100,and Triton X-114 are 14.9, 15.0, 13.4, and 12.3, respectively.Compared with other surfactants, using Triton-X-100 resultedin higher EFs for five of six compounds, especially for BEN andTEB, which had lower extraction efficiency. On the basis of theexperimental results, Triton X-100, which had better extractionefficiency and precision for most fungicides, was selected as thedispersed solvent.

Volume of Extraction Solvent. Different volumes of theextraction solvent (12, 14, 16, and 18 μL) were tested. Whenthe solvent volume was <12 μL, the amount of organic phasewas insufficient for analysis. As the volume of the extractionsolvent increased, the volume of the floating phase increasedaccordingly. This resulted in dilution of the extractants. TheEFs were maximal when the solvent volume was 12 μL. Hence,12 μL was selected as the volume of extraction solvent.

Concentration of Surfactant. The concentration ofsurfactant in aqueous solution plays an important role in effectiveextraction. The influence of the Triton X-100 concentration wasinvestigated by studying the results obtained with concentrationsof 1, 5, 10, and 50 mg L−1. At >50 mg L−1 surfactant, the solu-tion was still an emulsion after centrifugation. The floating phasecould not be collected. Therefore, the maximum concentrationof surfactant used was 50 mg L−1. When the concentration of

Figure 2. Effect of the type of surfactant for the first extraction solvent(n = 3). Samples were spiked with 5 μg L−1 of each analyte. Extractionconditions: extraction solvent, 1-octanol; volume, 12 μL; dispersedsolvent volume, 240 μL; concentration of the surfactant, 1 mg L−1.

Figure 3. Effect of the volume of the surfactant (n = 3). Samples were spiked with 5 μg L−1 of each analyte. Extraction conditions: extraction solvent,1-octanol; volume, 12 μL; dispersed solvent, Triton X-100; concentration, 10 mg L−1.

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surfactant in the aqueous solution was increased from 0 to10 mg L−1, the extraction EF was significantly improved.However, EFs declined slightly upon spiking with >10 mg L−1

Triton X-100. Evidently, higher concentrations of Triton X-100increased the solubility of analytes in the aqueous solutions,resulting in lower EFs. The optimal surfactant concentration inthe aqueous solution was therefore 10 mg L−1.Volume of the Surfactant Solution. To investigate the

impact of the volume of the aqueous solution, different volumesof aqueous solution (60, 120, 240, and 360 μL) containing10 mg L−1 Triton X-100 were evaluated (Figure 3). When thevolume of aqueous solution was changed from 60 to 240 μL,the EFs were increased. On the other hand, the EFs decreasedwhen the volume of aqueous solution was increased from240 to 360 μL. Therefore, the volume of aqueous solution wasfixed at 240 μL. The surfactant concentration was approx-imately equivalent to 0.48 mg L−1 in the total volume (5 mL) ofthe sample solution.Quantitative Aspects. The linearity obtained by using

12 μL of 1-octanol was evaluated under optimum conditions.Calibration curves were constructed by analyzing solutions ofwhite and red wines with DI water (1:1, v/v) at concentrationsof 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 μg L−1. Curves for allfungicides have coefficients of determination (R2) >0.9977.Data on the linear range (LR), R2, LOD, limit of quantification

(LOQ), precision (as relative standard deviation, RSD), and EFare summarized in Table 2. R2 values of the calibration curvesare in the range of 0.9977−0.9999, indicating high linearitywithin the concentration range used for each analyte. TheLODs were calculated as 3 times the standard deviation ofseven replicate runs of blank white and red wines spiked withlow concentrations of the analytes. LODs for determination offungicides in the blank wine samples differed substantially andranged from 0.013 to 0.155 μg L−1. Intra- and interday RSDsfor determination of the analytes ranged from 0.9 to 12.7% andfrom 3.4 to 14.1%, respectively.

Application in the Analysis of Wine Samples. Todemonstrate the capability of WLSSAE, the procedure wasapplied to the analysis of fungicides in white wine and red winesamples. The results show that the analyzed samples were freeof fungicides (Figure 4). FLU, BEN, and TEB have a broadshoulder (Figure 4b), which might result from the surfactantpartially extracted into the 1-octanol phase or from matrix effectof the samples. The wine samples were spiked with the analytesat 0.5, 5, and 50 μg L−1 levels. The absolute recovery (AR) wasdefined as the percentage of the total analyte (n0) that wasextracted into the floating organic phase (norg).

= ×n nAR / 100org 0

Table 2. Linearity, EFs, LODs, and Precision of Blank White and Red Wines

linearitya(n = 3, μg L−1) R2 EFsb LODc (μg L−1) LOQd (μg L−1)RSDe (%)

intraday, n = 7RSDe (%)

interday, n = 6

compd white red white red white red white red white red white red white red

CYP 0.05−100 0.05−100 0.9996 0.9997 414 349 0.021 0.036 0.072 0.121 7.2 2.3 11.2 3.3PRC 0.05−100 0.10−100 0.9999 0.9998 430 328 0.018 0.092 0.061 0.306 6.1 8.9 10.1 14.1FLD 0.1−100 0.05−100 0.9978 0.9977 1313 796 0.026 0.035 0.088 0.116 8.8 1.7 9.3 3.4FLU 0.05−100 0.05−100 0.9995 0.9984 904 505 0.013 0.020 0.044 0.069 4.4 0.9 4.3 5.6BEN 0.1−100 0.05−100 0.9998 0.9999 659 416 0.020 0.051 0.066 0.173 6.6 5.4 9.7 12.6TEB 0.05−100 0.50−100 0.9999 0.9996 1210 911 0.016 0.155 0.053 0.517 5.3 12.7 7.9 13.6

aBlank white and red wine samples spiked with 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 μg L−1. bBlank white and red wine samples spiked with 5 μg L−1,n = 3. cLODs were determined as 3 times the standard deviation obtained from seven replicate runs of blank white wine samples spiked with0.1 μg L−1 of each fungicide. dLOQs were determined as 10 times the standard deviation obtained from seven replicate runs of blank white and redwine samples spiked with 0.1 μg L−1 of each fungicide. eBlank wine samples were spiked with 0.5 μg L−1.

Figure 4. GC-MS selected ion chromatograms for the analysis of (a) white wine sample and (b) sample spiked with 5 μg L−1 fungicides: IS,ANT-d10; 1, CYP; 2, PRC; 3, FLD; 4, FLU; 5, BEN; 6, TEB.

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ARs were between 30 and 83% for white wine and between30 and 92% for red wine. The relative recovery (RR) is definedby the following equation:

= − ×C C CRR [( )/ ] 100found real added

Cfound, Creal, and Cadded are defined as the concentration ofanalyte after addition of a known amount of standard in the realsample, the concentration of analyte in the real sample, and theconcentration of known amount of standard that was spiked tothe real sample, respectively. RRs were between 81 and 108%for white wine and between 81 and 110% for red wine. RSDsfor the determination of the analytes ranged from 1.0 to 8.9%for white wine and from 1.0 to 7.2% for red wine (Table 3).This shows that after the wine samples were diluted with DIwater there was no significant matrix effect using WLSSAE.In Table 4, the performance of WLSSAE is compared with

that of other extraction techniques for the determination of

fungicides in wine. The results indicate that the proposedmethod is sensitive and applicable to the analysis of fungicidesat trace levels. The method requires a very short extractiontime (a few seconds) and 12 μL of extraction solvent only, incontrast to other methods.To extract fungicides from wine samples through the ISCS

technique, 0.48 mg L−1 of surfactants was required in using waterwith low concentration of surfactant as dispersed solvent. Theresults suggest that high EFs were achieved in a few seconds. Theapproach afforded high repeatability and high recovery within ashort extraction time. WLSSAE employed a few microliters of low-toxicity, halogen-free organic solvents to extract fungicides in winesamples. The analysis had a wide linear range and low LODs, aswell as high precision, EFs, and RRs. The LODs were much lowerthan the tolerance of procymidone in wine prescribed by theFDA.16 The method is a desirable application for the separationand preconcentration of fungicides at trace levels in wine samples.

Table 3. Accuracy, Absolute Recoveries (AR), Relative Recoveries (RR), and Precision (RSD) of Fungicides in Spiked Whiteand Red Wines

white wine (n = 3) red wine (n = 3)

WLSSAE spiked concn (μg L−1) calcd concn (μg L−1) AR (%) RR (%) RSD (%) calcd concn (μg L−1) AR (%) RR (%) RSD (%)

CYP 0.5 0.44 52 88 4.2 0.48 56 96 4.25 4.7 30 94 3.6 5.1 32 101 4.350 44.1 34 88 1.9 47.0 37 94 7.1

PRC 0.5 0.47 43 93 6.6 0.44 40 88 1.95 4.3 32 86 2.2 4.1 30 81 4.250 45.5 37 91 2.7 45.0 37 90 4.2

FLD 0.5 0.45 76 90 5.9 0.55 92 109 2.65 6.2 81 120 2.4 5.5 75 110 3.050 54.3 81 108 3.2 52.0 78 104 1.0

FLU 0.5 0.47 78 94 2.7 0.42 69 84 7.25 4.3 63 86 5.8 4.2 61 84 4.650 42.4 64 84 2.1 41.0 62 82 2.2

BEN 0.5 0.49 54 97 1.0 0.44 49 87 6.35 4.1 34 81 8.9 4.2 35 83 4.350 42.2 38 84 1.6 41.0 37 82 5.8

TEB 0.5 0.52 83 104 4.1 0.54 85 107 2.55 5.1 67 102 5.4 4.9 64 97 4.950 43.5 72 87 2.6 43.5 72 87 4.3

Table 4. Comparison of WLSSAE with Other Methods for Analysis of Fungicides in Wine

separation/detectionmethods sample extraction solvent (μL) dispersive agent (μL)

extraction time(min)

linear range(μg L−1) LOD (μg L−1) ref

SPE/LC-MSa wine 20 0.2−2000 0.06−0.51 24BAμE-LD/GC-MSb water/wine methanol (750) +

acetonitrile (750)255 0.04−1.6 0.004−0.030 25

DLLME-SFO/GC-MSc

wine 1-undecanol (50) acetone (500) 1 1−300 0.06−0.90 17

UASEME-SFO/HPLC-DADd

water/wine 1-undecanol (30) Tween 80 (10 mmol L−1,24 μL)

1 5−1000 1.2−4.7 38

WLSSAEe wine 1-octanol (12) Triton X-100 (10 mg L−1,240 μL)

few seconds 0.05−100 0.013−0.026 thiswork

aSolid phase extraction/liquid chromatography quadrupole time-of-flight tandem mass spectrometry. bBar adsorptive microextraction combinedwith liquid desorption/gas chromatography−mass spectrometry. cDispersive liquid−liquid microextraction based on solidification of floating organicdrop/gas chromatography−mass spectrometry. dUltrasound-assisted surfactant-enhanced emulsification microextraction/high-performance liquidchromatography with diode array detection. eWater with low concentration of surfactant in dispersed solvent-assisted emulsion dispersive liquid−liquid microextraction.

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■ AUTHOR INFORMATIONCorresponding Authors*(P.-S.C.) Phone: +886-2-2312-3456, ext. 65495. E-mail:paishanchen@ntu.edu.tw.*(S.-D.H.) Phone:+886-937997973. E-mail: sdhuang@mx.nthu.edu.tw.

Author Contributions∥W.-C.T and S.-P.C. contributed equally to this work.

FundingThis study was supported by the Ministry of Science andTechnology of Taiwan (NSC 99-2113-M-007-004-MY3).

NotesThe authors declare no competing financial interest.

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