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Jianzhi Sun1 Hui He1 Shuhui Liu1,2 1 College
of Science, Northwest A&F University, Yangling, China
2 State
Key Laboratory of Crop Stress Biology in Arid Areas, Yangling, China
Received February 1, 2014 Revised April 4, 2014 Accepted April 5, 2014
Research Article
Determination of phthalic acid esters in Chinese white spirit using dispersive liquid–liquid microextraction coupled with sweeping -cyclodextrin-modified micellar electrokinetic chromatography A simple method that consumes low organic solvent is proposed for the analysis of phthalic acid esters in Chinese white spirit using dispersive liquid–liquid microextraction coupled with sweeping-micellar electrokinetic chromatography. Tetrachloromethane and white-spirit-containing ethanol were used as the extraction and dispersing solvents, respectively. The electrophoresis separation buffer was composed of 5 mM -cyclodextrin, 50 mM sodium dodecyl sulfate and 25 mM borate buffer (pH 9.2) with 9% acetonitrile, enabling the baseline resolution of the analytes within 13 min. Under the optimum conditions, satisfactory linearities (5–1000 ng/mL, r 0.9909), good reproducibility (RSD 6.7% for peak area, and RSD 2.8% for migration time), low detection limits (0.4–0.8 ng/mL) and acceptable recovery rates (89.6–105.7%) were obtained. The proposed method was successfully applied to 22 Chinese white spirits, and the content of dibutyl phthalate in 55% of the samples exceeded the Specific Migration Limit of 0.3 mg/kg established by the domestic and international regulations. Keywords: Chinese White Spirit / Dispersive liquid-liquid microextraction / Micellar electrokinetic chromatography / Phthalic acid esters / Sweeping DOI 10.1002/jssc.201400118
1 Introduction Phthalic acid esters (PAEs) are used extensively as plasticizers for polymers including polyethylene (PE), polyethylene terephthalate (PET), polyvinyl acetates (PVA) and polyvinyl chloride (PVC), which aims at increasing their flexibility, extensibility and durability. Since PAEs are physically bound to the polymer structure, they can easily leach into the surrounding medium, consequently being ubiquitously found in the environment, house dust [1], various foods and alcohol products [2–4]. Since 1970s, extensive studies have examined the toxicity of PAEs. Dibutyl phthalate (DBP) and benzylbutyl phthalate (BBP) were found to be weakly estrogenic [5], and some PAEs are embryo fetal toxicants [6]. Owing to their potential risks to human health, the European Union established specific Correspondence: Dr. Shuhui Liu, College of Science, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shannxi 712100, China E-mail: [email protected] Fax: +86 29 87092226
Abbreviations: BBP, benzylbutyl phthalate; -CD-MEKC, -cyclodextrin-modified micellar electrokinetic chromatography; DBP, dibutyl phthalate; DEP, diethyl phthalate; DIBP, diisobutyl phthalate; DLLME, dispersive liquid–liquid microextraction; PAE, phthalic acid ester; SDS, sodium dodecyl sulfate; SML, specific migration limit
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
migration limits (SMLs) for DBP of 0.3 mg/kg and BBP of 30 mg/kg in the Directive 2007/19/EC relating to plastic materials and articles intended to come into contact with food [7], and the same SMLs for DBP and BBP were set in Chinese national standards GB/T9685-2008 [8]. In addition, the tolerable daily intakes of DBP [9] and BBP [10] specified by the European Food Safety Authority (EFSA) are 0.01 and 0.5 mg/kg body weight, respectively. The reference dose level (RfD) of diethyl phthalate (DEP) [11] derived by the United States Environment Protection Agency is 0.8 mg/kg body weight/day. Although inhalatory, dermal and endovenous routes of exposure to PAEs for humans are significant sources, the predominant one might be PAE-contaminated foods and drinks. Especially, owing to the lipophilic property of PAEs, they tend to migrate easily from plastic materials into fatty foods and alcohol products with high alcohol contents during production, transport and storage. GC is the chromatographic instrumentation most used for the analysis of PAEs, followed by HPLC, while CE has been used much less [12, 13]. The poor concentration sensitivity of CE with a UV detector can be offset by using online concentration strategies including field amplification, dynamic pH junction, transient isotachophoresis (tITP) and sweeping [14]. Sweeping is an effective on-line concentration technique for MEKC. It consists of the introduction of a large sample zone prepared in a matrix devoid of pseudostationary phase, wherein the analytes are picked up and accumulated by
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the pseudostationary phase that penetrates the sample zone and “sweeps” the analytes, producing a focusing effect. Prior to the instrumental determination of trace amount of PAEs in Chinese white spirit, extraction and preconcentration of the target analytes are required. LLE [15] and SPE [2] are conventionally used for the PAEs assay in wine, the advantages and disadvantages of which were stated in a number of related reports [2, 4, 15]. SPME is a relatively new microextraction approach with minimized organic solvent consumption and sample requirements, and automation capability [3]. Since dispersive liquid–liquid microextraction (DLLME) was introduced by Rezaee et al. in 2006 [16], it has gained popularity for its easy operation, low cost, short extraction time and high enrichment ratio. Recently, different DLLME modes, such as magnetic stirring-assisted DLLME (MSA-DLLME) [17], ultrasoundvortex-assisted DLLME (USVA-DLLME) [4] and ionic-liquidbased DLLME (IL-DLLME) [18], were proposed to extract PAEs in aqueous matrices. The separation techniques coupled to DLLME are mainly GC and HPLC. Because CE and DLLME have one common advantage of consuming very small volume of organic solvents, their combination is a much better choice than DLLME–HPLC in terms of green analytical chemistry. DLLME–CE without on-line concentration was applied to the analysis of phenolic compounds in aqueous cosmetics [19], sulfonamides in water [20], and phenolic acids in vegetable oils [21], achieving 0.025–10, 0.5–50 and 0.1–30 g/mL linear ranges, respectively. DLLME–CE with sweeping was adopted to analyze 5-nitroimidazole in water [22], carbamates in juice samples [23], achieving a linear range of 2.8–90 and 4–1000 g/L, respectively, comparable to DLLME–HPLC– UV. Despite these applications of DLLME–CE, the potential of DLLME has not been exploited as a sample treatment technique prior to CE. Herein, the objective of this study was to establish a simple and low organic solvent consuming analytical procedure for the determination of DEP, diisobutyl phthalate (DIBP), DBP and BBP in Chinese white spirit by combining DLLME and sweeping -cyclodextrin-modified micellar electrokinetic chromatography (sweeping--CD-MEKC). The effect of several factors that influenced performance of the microextraction including the type and volume of the extraction solvents, the volume of the disperser, and salt addition, were investigated and optimized. Sweeping effect was examined under both acidic and basic conditions. Twenty-two Chinese white spirits were assayed by the proposed procedure.
2 Materials and methods 2.1 Reagents and standards DEP (99.5%), DIBP (99%), DBP (99%) and BBP (99%) were purchased from Aladdin Chemistry (Shanghai, China). All chemicals used in the analysis were analytical grade. Na2 B4 O7 ·10H2 O, -cyclodextrin (-CD) (>99%) and NaCl C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
were purchased from BODI Chemical Reagent (Tianjin, China). NaH2 PO4 , H3 PO4 , HCl and NaOH were purchased from Xilong Chemical Reagent (Shantou, Guangdong Province, China). HPLC-grade CH3 OH, CH3 CN, CCl4 , CHCl3 , CH2 Cl2 and C6 H5 Cl were obtained from Kermel Chemical Reagent (Tianjin, China). Sodium dodecyl sulfate (SDS) was provided by Sanland (Los Angeles, CA, USA). Stock standard solutions of each PAE were prepared at 2 mg/mL in CH3 OH and stored at –10⬚C in darkness. Working standard solutions were prepared daily by suitable dilutions.
2.2 Apparatus The quantitative analysis was performed on a Beckman P/ACETM MDQ Capillary Electrophoresis System equipped with an autosampler and a diode array detector (DAD). All of the operations were computer-controlled using Beckman P/ACE MDQ 32 karat software. Uncoated fused-silica capillaries, 75 m × 50.2 cm (40 cm to the detector), from Rui feng Optical Fiber Factory (Yongnian, Hebei Province, China), were maintained at 25⬚C in a cartridge. The DAD was set at an acquisition range from 190 to 400 nm (bandwidth of 6 nm) at a spectral acquisition rate of 4 Hz. The detection wavelength used was 206 nm. The pH of all solutions was measured by pH 213 equipment (Hanna instruments, Italy). A Model KQ-5200E ultrasonic cleaning machine from Kunshan City Ultrasonic Instruments (Kunshan, Jiangsu Province, China) was used. Centrifugation was done with a Beckman Allegra X-12 system from Beckman Coulter (Fullerton, USA). Water was purified using a Millipore Direct-Q 3 system (Millipore, Bedford, MA, USA). Before the first use, the new capillary was rinsed sequentially with CH3 OH (5 min), water (5 min), 0.1 M HCl (10 min), water (5 min), 0.1 M NaOH (30 min) and water (5 min), finally the BGE (20 min). To ensure repeatability, the capillary was flushed between consecutive analyses with 0.1 M NaOH (3 min), water (2 min), finally the running buffer for 5 min. In all instances, a pressure of 20 psi was applied. To prevent capillary blockage, the buffer was degassed by sonication and filtered through 0.22 m filter membranes from Shanghai Minimo Separation Technology (Shanghai, China).
2.3 Sample and sample preparation Twenty-two Chinese white spirit samples (numbered from S-1 to S-22) were purchased from the local supermarkets, in which the alcohol contents were in the range of 45–54% v/v. A mixture of 100 L CCl4 and 2 mL Chinese white spirit was rapidly injected into 8 mL NaCl solution (containing 1 g NaCl). After shaking for 3 min, the mixture was centrifuged at 4000 rpm for 3 min. The sedimented phase found at the bottom of the tube was transferred to a glass vial using a microsyringe. The organic phase was evaporated at 30⬚C under www.jss-journal.com
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nitrogen stream until dryness and then re-suspended with 0.5 mL 80 mM borate solution containing 10% v/v CH3 OH for CE analysis.
2.4 Sweeping Before sample injection, the capillary was conditioned with -CD modified micellar buffer solution consisted of 5 mM -CD, 50 mM SDS, 25 mM borate buffer (pH 9.2 adjusted with 2 M NaOH) containing 9% v/v CH3 CN. Then the large plug of sample was hydrodynamically injected for 15 s at 3 psi. Afterwards, with the inlet of the capillary switched to the BGE vial, a high voltage (22 kV) was then applied for both sample sweeping and subsequent separation.
2.5 Glassware and blank control To avoid exogenous PAEs contamination, all glassware used in the study were soaked in acetone for at least 30 min, next rinsed with acetone followed by hexane, and finally dried at 120⬚C for at least 4 h. Three blanks were prepared per extraction batch by using 2 mL water in place of the Chinese white spirit sample. The concentrations of PAEs found in the blanks were averaged and subtracted from sample assay results.
3 Results and discussion 3.1 Development of sweeping-MEKC method Sweeping-MEKC can be performed with both acidic and basic buffers. Quirino et al. [24] studied the relationship between sweeping efficiency and the buffer pH, indicating the peak widths of alkyl phenyl ketones achieved with an acidic BGE were much narrower than with a basic one. The results implied that the theoretical sample injection length with the former should be much longer than with the latter, which has been verified by a number of subsequent reports. For instance, under acidic sweeping-MEKC conditions, the injection length was 41.16 cm for linezolid [25], 10.9 cm for neonicotinoid insecticides [26] and 15 cm for steroids [27]; however, for alkaline sweeping-MEKC, 5.52 cm for all transand 13 cis-retinoic acids [28], 4.66 cm for neutral steroids [29] and 2.45 cm for aflatoxins [30]. In this study, both acidic and basic BGEs were investigated and optimized, and the results obtained were compared. In preliminary experiments, the baseline separation could not be achieved in an acidic BGE with an amount of CH3 CN < 20% v/v. Different contents of CH3 CN (25– 35%) were initially investigated in conjunction with the buffer containing 25 mM NaH2 PO4 and 50 mM SDS (pH 2.3 adjusted with 3 M H3 PO4 ). And the results showed that 30% CH3 CN was optimal. Subsequently, the concentrations of the NaH2 PO4 buffer (10–30 mM) and SDS (30–60 mM) were ex C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Effect of -CD concentration in BGE. Experimental conditions: BGE, 55 mM SDS + 10% v/v CH3 CN + 25 mM borate buffer (pH 9.2); other conditions: 28 kV; sample matrix, 30 mM borate buffer (pH 9.2, containing 10% v/v CH3 OH); injection length, 17.4 cm. Concentration of each PAE: 0.5 g/mL; Peaks: 1, DEP; 2, DIBP; 3, DBP; 4, BBP.
amined, and 10 mM NaH2 PO4 and 40 mM SDS were adopted. To improve the resolution between BBP and DBP, -CD (0–15 mM) was added into the buffer due to its powerful separation capacity for lipophilic compounds with similar structures, but the results showed no improvement was achieved. It could be speculated that CH3 CN was functioning as a competitive substance; as its concentration was increased, to a certain extent it effectively competed to displace the target analytes from the guest cavity of -CD [31], which restrained the capacity of -CD partitioning PAEs. The NaH2 PO4 content varying from 10 to 30 mM in the sample solution containing 10% v/v CH3 OH was investigated, and the result indicated it had no positive influence on the focusing effect and resolution. Sample zone length injected ranging from 8.7 to 34.8 cm was examined, and the results demonstrated broaden bands and poor resolution occurred with the length exceeding 18 cm. Eventually, a sample matrix of 10 mM NaH2 PO4 and an injection length of 17.4 cm were used. The sweeping--CD-MEKC method under alkaline BGE was studied. Firstly, the -CD concentration (0–10 mM) was modified with the buffer consisting of 25 mM borate buffer and 55 mM SDS (pH 9.2) containing 10% v/v CH3 CN. As shown in Fig. 1, both resolution and sensitivity were poor for DIBP, DBP and BBP without incorporation of -CD, but 5 mM -CD gave good resolutions, which was used in subsequent investigations. The concentration of borate buffer concentration (15– 30 mM) and the pH value (8.5–10.0) were tested. The complete separation was obtained at 25 mM borate with a pH value of 9.2. The SDS concentrations (50–80 mM) were also studied, and results showed that 50 mM SDS was the optimal value. 9% v/v CH3 CN was used to further improve the separation. www.jss-journal.com
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A
Figure 2. Effect of borate concentration in sample matrix. Experimental conditions: BGE, 5 mM -CD + 50 mM SDS + 9% v/v CH3 CN + 25 mM borate buffer (pH 9.2); Concentration of each PAE: 2.0 g/mL; other conditions: 22 kV; injection length, 13 cm.
The effect of the conductivity in the sample matrix on the focus effect was examined by dissolving the analytes (2.0 g/mL) in the buffers containing 30–80 mM borate (pH 9.2) with 10% v/v CH3 OH. As shown in Fig. 2, when increasing the borate content from 30 to 80 mM, the focus effect was improved and the peak shapes tended to be better. A sample matrix with higher conductivity (e.g. 80 mM borate buffer) produced a reduced electric field in sample zone and an enhanced electric field in BGE region. When voltage was applied, SDS stacked in the front of high conductivity sample zone. In this case, the concentration of SDS entering the sample zone was higher than under a homogenous electric field or an enhanced electric field in the sample zone. Hence, the analytes were picked up and accumulated by the stacked SDS penetrating the sample zone, and the final swept zone was narrower. Therefore, 80 mM borate buffer was selected. Sample injection length was investigated from 8.7 to 26.1 cm. The results demonstrated that increasing the injection length decreased the migration time, and the resolutions and peak shapes were gradually deteriorated with the length of injected sample > 13 cm. As a result, a sample plug of 13 cm was chosen in this study. Figure 3 showed typical CE electropherograms of PAEs standard obtained in the acidic (Fig. 3A) and alkaline buffers (Fig. 3B), respectively. As illustrated, although slightly better enrichment effect was achieved with the acidic BGE, the basic BGE provided a much shorter migration time and baseline resolution, which was eventually selected.
3.2 Optimization of DLLME conditions In the previously reported DLLME procedures for the analysis of PAEs in water samples, the extractant mixture used were CCl4 /CH3 CN [32] and C6 H5 Cl/acetone [33]. In this study, as C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B
Figure 3. Typical electropherograms of PAEs standard (1 g/mL) by the acidic (A) and alkaline separation conditions (B). Experimental conditions: (A), BGE, 40 mM SDS +30% v/v CH3 CN + 10 mM NaH2 PO4 (pH 2.3); other conditions, –22 kV; injection length, 17.4 cm. (B), BGE was the same as Fig. 2. Peaks of Fig. 3A: 1, BBP; 2, DBP; 3, DIBP; 4, DEP.
Chinese white spirit samples usually contain about 50% v/v ethanol, which can dissolve most of DLLME extractants, no additional disperser was therefore required. In this context, the effects of various experimental parameters, such as the type and volume of extraction solvents, the disperser volume and salt addition, on DLLME efficiency were optimized. In the preliminary experiment, it was found that if adopting 20% v/v ethanol as the disperser, there was scarcely any sedimented phase after microextraction and centrifugation. Therefore, 2 mL Chinese white spirit sample mixed with 8 mL 10% v/v NaCl solution was used to optimize the parameters. 120 L of different extraction solvents including CCl4 (1.59 g/mL), CHCl3 (1.47 g/mL), CH2 Cl2 (1.32 g/mL) and C6 H5 Cl (1.10 g/mL) were tested. When using CH2 Cl2 , the sedimented phase could not form, which could be due to its comparatively high solubility in water (the solubility of CH2 Cl2 , CHCl3 , CCl4 and C6 H5 Cl at 20⬚C in water are 20, 8, 0.8 and 0.49 g/L, respectively). C6 H5 Cl and CHCl3 provided low extraction recoveries% (ERs%) for DBP and www.jss-journal.com
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Table 1. Linearity, calibration curve, r, LODs, LOQs, EF and repeatability data obtained for a spiked Chinese white spirit sample
Linearity (ng/mL) Calibration graphs Calibration curve (n = 7) r LODs (ng/mL) LOQs (ng/mL) Enhancement factor (EF) DLLME Sweeping--CD-MEKC DLLME-sweeping--CD-MEKC Reproducibility (RSD%) Peak area 20 ng/mL 100 ng/mL 300 ng/mL Migration time 20 ng/mL 100 ng/mL 300 ng/mL
DEP
DIBP
DBP
BBP
10500
51000
51000
5800
Y = 101.83X + 1070.2 0.9909 0.8 2.7
Y = 98.96X + 828.58 0.9988 0.6 1.9
Y = 86.45X + 722.26 0.9969 0.5 1.6
Y = 104.59X + 618.17 0.9990 0.4 1.4
4 25 92
3 25 81
3 24 79
3 26 82
5.7 3.5 2.9
3.7 4.8 3.7
5.9 4.3 2.5
6.7 4.8 4.9
2.1 1.6 1.2
1.6 1.3 2.8
2.1 1.7 1.9
1.9 2.3 2.0
DIBP. Among these extraction solvents, CCl4 gave the highest ERs% for all the target analytes, thus it was selected for subsequent experiments. To illustrate the effect of the disperser volume on ERs%, the different volumes of Chinese white spirit (1–4 mL) were diluted to the bulk volume of 10 mL with aqueous NaCl solution, which produced the sample matrices containing 5, 10, 15, and 20% v/v ethanol, respectively. The results showed that the best ERs% of PAEs was obtained when using 2 mL Chinese white spirit sample. The volume of CCl4 was tested in the range of 60– 140 L in 20 L intervals. It was observed that the ERs% rapidly increased from 60 to 100 L, after which it remained constant until the CCl4 volume was increased up to 140 L. On the basis of this result, 100 L CCl4 was utilized for the following experiments. The different concentrations of NaCl ranging from 0 to 20% w/v were evaluated. The results showed that, increasing the salt content from 0 to 10% w/v, the ERs% of each analyte increased gradually; with the amount of NaCl exceeding 10% w/v, the ERs% varied negligibly. It could be explained that ERs% increased due to salting out, whereby water molecules form hydration spheres around the ionic salt molecules that reduce the concentration of water available to dissolve the analytes molecules, thereby driving the additional analytes into the organic droplets [19]. Therefore, 10% w/v NaCl was used. In summary, the optimal extraction procedure was performed by adding a mixture of 100 L CCl4 and 2 mL Chinese white spirit to 8 mL salt solution (containing 1 g NaCl), followed by a manual shaking time of 3 min and centrifugation time of 3 min at 4000 rpm. The sedimented phase was evaporated at 30⬚C under nitrogen stream until dryness and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
re-suspended with 0.5 mL 80 mM borax containing 10% v/v CH3 OH.
3.3 Method validation and comparison with other analytical procedures Under optimum conditions, method performance was evaluated in terms of linearity, LODs, LOQs, enhancement factors (EFs) and reproducibility. Good linearity was observed for all the analytes, and the regression equations were shown in Table 1. The LODs (S/N = 3) ranged from 0.4 to 0.8 ng/mL, and the LOQs (S/N = 10) were between 1.4 and 2.7 ng/mL. The EFs with DLLME-sweeping--CD-MEKC as compared to conventional MEKC were in the range of 79- to 92-fold. As can be seen in Table 1, the results showed acceptable reproducibility in all cases with RSD% values for peak area and migration time less than 6.7 and 2.8%, respectively. As shown in Table 2, the recoveries of the four PAEs were in the range of 89.6–105.7% with RSDs% less than 6.3%. Table 3 showed the comparison of the proposed approach and other reported methods by virtue of organic solvent consumption, extraction time, LOQs and EFs. The volume of organic solvent consumed in this method (100 L CCl4 ) was much less than the SPE-MEKC (29 mL CH3 OH) [12]; the LOQs obtained in this study (1.4–2.7 ng/mL) were lower than the LOQs of the IL-DLLME (10.6–28.5 ng/mL) [18], sweeping-MEEKC (75–150 ng/mL) [34] and SPE-MEKC (21400–57700 ng/mL) [12]. Although the USVA-DLLME–GC with flame ionization detection (FID) and ion trap mass spectrometry (ITMS) procedure consumed less extraction solvent (40 L CH2 Cl2 ) [4] and provided lower LOQs www.jss-journal.com
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Table 2. Spiked recoveries of PAEs in real samples by the proposed method (n = 3)
Samples
Added (ng/mL)
DEP Recovery% (RSD%)
DIBP Recovery% (RSD%)
DBP Recovery% (RSD%)
BBP Recovery% (RSD%)
S-2
50 150 300 20 150 300 50 150 300
92.8 (2.6) 96.4 (2.4) 94.7 (3.1) 92.7 (2.6) 94.6 (4.2) 92.1 (2.2) 92.7 (2.6) 95.3 (2.4) 95.9 (3.0)
92.3 (4.7) 93.7 (1.2) 91.9 (3.4) 90.5 (2.0) 105.7 (5.5) 91.4 (3.1) 92.4 (3.9) 94.1 (2.8) 90.4 (1.2)
91.5 (4.7) 92.6 (6.3) 89.8 (3.5) 95.3 (1.9) 93.1 (2.9) 90.7 (1.4) 93.5 (4.0) 95.2 (2.3) 92.3 (1.7)
90.9 (1.2) 94.6 (5.3) 91.6 (3.1) 90.4 (1.3) 94.9 (2.9) 89.6 (3.8) 91.2 (5.6) 94.5 (4.2) 91.3 (3.2)
S-5
S-10
Table 3. Comparison of the current work with other previously reported methods for the determination of PAEs
Method
Sample volume Matrix
Organic solvent consumption
Extraction time (min)
LOQs (ng/mL)
RSD%
Enrichment factor
Ref
10.6–28.5
7.8–15
—
[18]
0.4–1.3
1.5–3.4
14-49
[17]
0.08–0.4
4.9–8.2
220-300
[4]
IL-DLLME–HPLC–UV a) 10 mL
Water
MSA-DLLME–HPLC– UV b) USVA-DLLME–GC–FID/ ITMS c) Sweeping-MEEKC– DAD SPE–MEKC–DAD DLLME-sweeping-CD-MEKC–DAD
10 mL
Drinking+water
10 mL
Wine
Acetone (1 mL) 15 + [C8 MIM][PF6 ] (80 L) Dodecane 10 (200 L) CH2 Cl2 (40 L) 15
—
Soft drinks
—
—
75–150
4.9
—
[34]
500 L 2 mL
Perfume Chinese white spirit
CH3 OH (29 mL) CCl4 (100 L)
20 15
21,400–57,700 1.4–2.7
2.5–11.6 2.5–6.7
— 79–92
[12] This method
a) Ionic liquid-based-DLLME–HPLC–UV. b) Magnetic stirring-assisted-DLLME–HPLC–UV. c) Ultrasound-vortex-assisted-DLLME–GC–FID/ITMS.
(0.08–0.4 ng/mL) than the current method, the preliminary experiment showed CH2 Cl2 could not present efficient extraction for PAEs in Chinese white spirits.
3.4 Assays of real Chinese white spirit samples The proposed DLLME-sweeping--CD-MEKC method was applied to determine the four PAEs in 22 Chinese white spirits. The typical electropherograms for S-5 (a) and spiked S-5 (b) and S-12 (c) are depicted in Fig. 4, and the assay data are shown in Fig. 5. As illustrated in Fig. 5, among the 22 Chinese white spirits, DEP and BBP were found in low levels and frequencies, but the detection frequencies for DIBP and DBP were 95.5 and 100%, respectively, which were also frequently found in previous works [2–4, 15]. The contents of DBP in 55% of the Chinese white spirit samples exceeded the established C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SML (0.3 mg/kg) in Directive 2007/19/EC, and the maximum (5.569 g/mL) was about 18.6 times with respect to the SML. Although it is difficult to give information about the source of DIBP or DBP in Chinese white spirit on the basis of the experimental results, it is worth noting that people should refrain from over-drinking Chinese white spirit and pay more attention to its possible PAEs contamination.
4 Conclusions A novel method based on DLLME coupled with sweeping-CD-MEKC was successfully developed for the determination of PAEs in Chinese white spirits. The presented method accomplished the baseline resolution among DIBP, DBP and BBP within 13 min, exhibiting significantly higher separation efficiency as compare with HPLC procedures. The combination of DLLME and sweeping-MEKC enabled the www.jss-journal.com
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5 References [1] Bornehag, C. G., Lundgren, B., Weschler, C. J., Sigsgaard, T., Hagerhed-Engman, L., Sundell, J., Environ. Health Perspet. 2005, 113, 1399–1404. [2] Carlo, M. D., Pepe, A., Sacchetti, G., Compagnone, D., Mastrocola, D., Cichelli, A., Food Chem. 2008, 111, 771–777. [3] Ye, C. W., Gao, J., Yang, C., Liu, X. J., Li, X. J., Pan, S. Y., Anal. Chim. Acta 2009, 641, 64–74. [4] Cinelli, G., Avino, P., Notardonato, I., Centola, A., Russo, M. V., Anal. Chim. Acta 2013, 769, 72–78. [5] Jobling, S., Reynolds, T., White, R., Parker, M. G., Sumpter, J. P., Environ. Health Perspect. 1995, 103, 582–587. [6] Ema, M., Kurosaka, R., Amano, H., Ogawa, Y., Reprod. Toxicol. 1994, 8, 231–236. Figure 4. Typical electropherograms for blank and spiked Chinese white spirit sample obtained under optimum extraction and sweeping conditions. (A) S-5; (B) S-5 spiked with 0.20 g/mL of each analyte; (C) S-12.
[7] Commission Directive 2007/19/EC, Off. J. Eur. Commun. 2007, L91, 17. [8] National Standards of the People’s Republic of China, Hygienic standards for uses of additives in food containers and packaging materials, China, 2008. [9] European Food Safety Authority, EFSA J. 2005, 242, 1–17. [10] European Food Safety Authority, EFSA J. 2005, 241, 1–14. [11] Heudorf, U., Mersch-Sundermann, V., Angerer, J., Int. J. Hyg. Environ. Health 2007, 210, 623–634. ´ Ma´ ´ ´ [12] Perez-Fern andez, V., Gonzalez, M. J., Garc´ıa, M. A., rina, M. L., Anal. Chim. Acta 2013, 782, 67–74. [13] Lin, Z., Zhang, J. H., Cui, H. M., Zhang, L., Chen, G. N., J. Sep. Sci. 2010, 33, 3717–3725. [14] Zhang, Y. H., Jiao, B. N., J. Sep. Sci. 2013, 36, 3067–3074. [15] Guo, Y., Zhang, Z. F., Liu, L. Y., Li, Y. F., Ren, N. Q., Kannan, K., J. Agric. Food Chem. 2012, 60, 6913–6919. [16] Rezaee, M., Assadi, Y., Hosseini, M. M., Aghaee, E., Ahmadi, F., Berijani, S., J. Chromatogr. A 2006, 1116, 1–9. [17] Ranjbari, E., Hadimohammadi, M. R., Talanta 2012, 100, 447–453.
Figure 5. Contents of DIBP, DBP and the sum of DIBP and DBP in 22 Chinese white spirit samples.
[18] Chen, S., Zhong, Y. S., Cheng, S. Y., Qian, T., Sun, H., J. Sep. Sci. 2011, 34, 1503–1507. [19] Zhou, C. H., Tong, S. S., Chang, Y. X., Jia, Q., Zhou, W. H., Electrophoresis 2012, 33, 1331–1338.
quantitative analysis of PAEs at ng/mL level, which was much lower than the SML (0.3 mg/kg) established by the European Union and the Chinese national standard. Moreover, the present procedure offered the advantages of simplicity, low consumption of toxic organic solvents and low cost. Equipped with the merits and features, this method could be exploited as an alternative routine analysis method for the PAEs analysis in aqueous matrix. The authors acknowledge with gratitude and appreciation financial support from Northwest A&F University. The authors have declared no conflict of interest. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[20] Wen, Y. Y., Li, J. H., Zhang, W. W., Chen, L. X., Electrophoresis 2011, 32, 2131–2138. [21] Bakar, N. B. A., Makahleh, A., Saad, B., Anal. Chim. Acta 2012, 742, 59–66. ´ [22] Hernandez-Mesa, M., Cruces-Blanco, C., Garc´ıa˜ A. M., J. Sep. Sci. 2013, 35, 3050–3058. Campana, ´ ´ [23] Moreno-Gonzalez, D., Gamiz-Gracia, L., Garc´ıa˜ A. M., Bosque-Sendra, J. M., Anal. Bioanal. Campana, Chem. 2011, 400, 1329–1338. [24] Quirino, J. P., Terabe, S., Anal. Chem. 1999, 71, 1638–1644. [25] Michalska, K., Pajchel, G., Tyski, S., J. Pham. Biomed. Anal. 2008, 48, 321–330. [26] Fang, H. F., Yang, F. X., Sun, J. L., Tian, Y., Zeng, Z. R., Xu, Y., Talanta 2011, 85, 2148–2153. www.jss-journal.com
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[31] Connors, K. A., Chem. Rev. 1997, 97, 1325–1357. [32] Liang, P., Xu, J., Li, Q., Anal. Chim. Acta 2008, 609, 53–58. [33] Farahani, jali, M. 105–112.
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Jianzhi Sun1 Hui He1 Shuhui Liu1,2 1 College
of Science, Northwest A&F University, Yangling, China
2 State
Key Laboratory of Crop Stress Biology in Arid Areas, Yangling, China
Received February 1, 2014 Revised April 4, 2014 Accepted April 5, 2014
Research Article
Determination of phthalic acid esters in Chinese white spirit using dispersive liquid–liquid microextraction coupled with sweeping -cyclodextrin-modified micellar electrokinetic chromatography A simple method that consumes low organic solvent is proposed for the analysis of phthalic acid esters in Chinese white spirit using dispersive liquid–liquid microextraction coupled with sweeping-micellar electrokinetic chromatography. Tetrachloromethane and white-spirit-containing ethanol were used as the extraction and dispersing solvents, respectively. The electrophoresis separation buffer was composed of 5 mM -cyclodextrin, 50 mM sodium dodecyl sulfate and 25 mM borate buffer (pH 9.2) with 9% acetonitrile, enabling the baseline resolution of the analytes within 13 min. Under the optimum conditions, satisfactory linearities (5–1000 ng/mL, r 0.9909), good reproducibility (RSD 6.7% for peak area, and RSD 2.8% for migration time), low detection limits (0.4–0.8 ng/mL) and acceptable recovery rates (89.6–105.7%) were obtained. The proposed method was successfully applied to 22 Chinese white spirits, and the content of dibutyl phthalate in 55% of the samples exceeded the Specific Migration Limit of 0.3 mg/kg established by the domestic and international regulations. Keywords: Chinese White Spirit / Dispersive liquid-liquid microextraction / Micellar electrokinetic chromatography / Phthalic acid esters / Sweeping DOI 10.1002/jssc.201400118
1 Introduction Phthalic acid esters (PAEs) are used extensively as plasticizers for polymers including polyethylene (PE), polyethylene terephthalate (PET), polyvinyl acetates (PVA) and polyvinyl chloride (PVC), which aims at increasing their flexibility, extensibility and durability. Since PAEs are physically bound to the polymer structure, they can easily leach into the surrounding medium, consequently being ubiquitously found in the environment, house dust [1], various foods and alcohol products [2–4]. Since 1970s, extensive studies have examined the toxicity of PAEs. Dibutyl phthalate (DBP) and benzylbutyl phthalate (BBP) were found to be weakly estrogenic [5], and some PAEs are embryo fetal toxicants [6]. Owing to their potential risks to human health, the European Union established specific Correspondence: Dr. Shuhui Liu, College of Science, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shannxi 712100, China E-mail: [email protected] Fax: +86 29 87092226
Abbreviations: BBP, benzylbutyl phthalate; -CD-MEKC, -cyclodextrin-modified micellar electrokinetic chromatography; DBP, dibutyl phthalate; DEP, diethyl phthalate; DIBP, diisobutyl phthalate; DLLME, dispersive liquid–liquid microextraction; PAE, phthalic acid ester; SDS, sodium dodecyl sulfate; SML, specific migration limit
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
migration limits (SMLs) for DBP of 0.3 mg/kg and BBP of 30 mg/kg in the Directive 2007/19/EC relating to plastic materials and articles intended to come into contact with food [7], and the same SMLs for DBP and BBP were set in Chinese national standards GB/T9685-2008 [8]. In addition, the tolerable daily intakes of DBP [9] and BBP [10] specified by the European Food Safety Authority (EFSA) are 0.01 and 0.5 mg/kg body weight, respectively. The reference dose level (RfD) of diethyl phthalate (DEP) [11] derived by the United States Environment Protection Agency is 0.8 mg/kg body weight/day. Although inhalatory, dermal and endovenous routes of exposure to PAEs for humans are significant sources, the predominant one might be PAE-contaminated foods and drinks. Especially, owing to the lipophilic property of PAEs, they tend to migrate easily from plastic materials into fatty foods and alcohol products with high alcohol contents during production, transport and storage. GC is the chromatographic instrumentation most used for the analysis of PAEs, followed by HPLC, while CE has been used much less [12, 13]. The poor concentration sensitivity of CE with a UV detector can be offset by using online concentration strategies including field amplification, dynamic pH junction, transient isotachophoresis (tITP) and sweeping [14]. Sweeping is an effective on-line concentration technique for MEKC. It consists of the introduction of a large sample zone prepared in a matrix devoid of pseudostationary phase, wherein the analytes are picked up and accumulated by
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the pseudostationary phase that penetrates the sample zone and “sweeps” the analytes, producing a focusing effect. Prior to the instrumental determination of trace amount of PAEs in Chinese white spirit, extraction and preconcentration of the target analytes are required. LLE [15] and SPE [2] are conventionally used for the PAEs assay in wine, the advantages and disadvantages of which were stated in a number of related reports [2, 4, 15]. SPME is a relatively new microextraction approach with minimized organic solvent consumption and sample requirements, and automation capability [3]. Since dispersive liquid–liquid microextraction (DLLME) was introduced by Rezaee et al. in 2006 [16], it has gained popularity for its easy operation, low cost, short extraction time and high enrichment ratio. Recently, different DLLME modes, such as magnetic stirring-assisted DLLME (MSA-DLLME) [17], ultrasoundvortex-assisted DLLME (USVA-DLLME) [4] and ionic-liquidbased DLLME (IL-DLLME) [18], were proposed to extract PAEs in aqueous matrices. The separation techniques coupled to DLLME are mainly GC and HPLC. Because CE and DLLME have one common advantage of consuming very small volume of organic solvents, their combination is a much better choice than DLLME–HPLC in terms of green analytical chemistry. DLLME–CE without on-line concentration was applied to the analysis of phenolic compounds in aqueous cosmetics [19], sulfonamides in water [20], and phenolic acids in vegetable oils [21], achieving 0.025–10, 0.5–50 and 0.1–30 g/mL linear ranges, respectively. DLLME–CE with sweeping was adopted to analyze 5-nitroimidazole in water [22], carbamates in juice samples [23], achieving a linear range of 2.8–90 and 4–1000 g/L, respectively, comparable to DLLME–HPLC– UV. Despite these applications of DLLME–CE, the potential of DLLME has not been exploited as a sample treatment technique prior to CE. Herein, the objective of this study was to establish a simple and low organic solvent consuming analytical procedure for the determination of DEP, diisobutyl phthalate (DIBP), DBP and BBP in Chinese white spirit by combining DLLME and sweeping -cyclodextrin-modified micellar electrokinetic chromatography (sweeping--CD-MEKC). The effect of several factors that influenced performance of the microextraction including the type and volume of the extraction solvents, the volume of the disperser, and salt addition, were investigated and optimized. Sweeping effect was examined under both acidic and basic conditions. Twenty-two Chinese white spirits were assayed by the proposed procedure.
2 Materials and methods 2.1 Reagents and standards DEP (99.5%), DIBP (99%), DBP (99%) and BBP (99%) were purchased from Aladdin Chemistry (Shanghai, China). All chemicals used in the analysis were analytical grade. Na2 B4 O7 ·10H2 O, -cyclodextrin (-CD) (>99%) and NaCl C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
were purchased from BODI Chemical Reagent (Tianjin, China). NaH2 PO4 , H3 PO4 , HCl and NaOH were purchased from Xilong Chemical Reagent (Shantou, Guangdong Province, China). HPLC-grade CH3 OH, CH3 CN, CCl4 , CHCl3 , CH2 Cl2 and C6 H5 Cl were obtained from Kermel Chemical Reagent (Tianjin, China). Sodium dodecyl sulfate (SDS) was provided by Sanland (Los Angeles, CA, USA). Stock standard solutions of each PAE were prepared at 2 mg/mL in CH3 OH and stored at –10⬚C in darkness. Working standard solutions were prepared daily by suitable dilutions.
2.2 Apparatus The quantitative analysis was performed on a Beckman P/ACETM MDQ Capillary Electrophoresis System equipped with an autosampler and a diode array detector (DAD). All of the operations were computer-controlled using Beckman P/ACE MDQ 32 karat software. Uncoated fused-silica capillaries, 75 m × 50.2 cm (40 cm to the detector), from Rui feng Optical Fiber Factory (Yongnian, Hebei Province, China), were maintained at 25⬚C in a cartridge. The DAD was set at an acquisition range from 190 to 400 nm (bandwidth of 6 nm) at a spectral acquisition rate of 4 Hz. The detection wavelength used was 206 nm. The pH of all solutions was measured by pH 213 equipment (Hanna instruments, Italy). A Model KQ-5200E ultrasonic cleaning machine from Kunshan City Ultrasonic Instruments (Kunshan, Jiangsu Province, China) was used. Centrifugation was done with a Beckman Allegra X-12 system from Beckman Coulter (Fullerton, USA). Water was purified using a Millipore Direct-Q 3 system (Millipore, Bedford, MA, USA). Before the first use, the new capillary was rinsed sequentially with CH3 OH (5 min), water (5 min), 0.1 M HCl (10 min), water (5 min), 0.1 M NaOH (30 min) and water (5 min), finally the BGE (20 min). To ensure repeatability, the capillary was flushed between consecutive analyses with 0.1 M NaOH (3 min), water (2 min), finally the running buffer for 5 min. In all instances, a pressure of 20 psi was applied. To prevent capillary blockage, the buffer was degassed by sonication and filtered through 0.22 m filter membranes from Shanghai Minimo Separation Technology (Shanghai, China).
2.3 Sample and sample preparation Twenty-two Chinese white spirit samples (numbered from S-1 to S-22) were purchased from the local supermarkets, in which the alcohol contents were in the range of 45–54% v/v. A mixture of 100 L CCl4 and 2 mL Chinese white spirit was rapidly injected into 8 mL NaCl solution (containing 1 g NaCl). After shaking for 3 min, the mixture was centrifuged at 4000 rpm for 3 min. The sedimented phase found at the bottom of the tube was transferred to a glass vial using a microsyringe. The organic phase was evaporated at 30⬚C under www.jss-journal.com
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nitrogen stream until dryness and then re-suspended with 0.5 mL 80 mM borate solution containing 10% v/v CH3 OH for CE analysis.
2.4 Sweeping Before sample injection, the capillary was conditioned with -CD modified micellar buffer solution consisted of 5 mM -CD, 50 mM SDS, 25 mM borate buffer (pH 9.2 adjusted with 2 M NaOH) containing 9% v/v CH3 CN. Then the large plug of sample was hydrodynamically injected for 15 s at 3 psi. Afterwards, with the inlet of the capillary switched to the BGE vial, a high voltage (22 kV) was then applied for both sample sweeping and subsequent separation.
2.5 Glassware and blank control To avoid exogenous PAEs contamination, all glassware used in the study were soaked in acetone for at least 30 min, next rinsed with acetone followed by hexane, and finally dried at 120⬚C for at least 4 h. Three blanks were prepared per extraction batch by using 2 mL water in place of the Chinese white spirit sample. The concentrations of PAEs found in the blanks were averaged and subtracted from sample assay results.
3 Results and discussion 3.1 Development of sweeping-MEKC method Sweeping-MEKC can be performed with both acidic and basic buffers. Quirino et al. [24] studied the relationship between sweeping efficiency and the buffer pH, indicating the peak widths of alkyl phenyl ketones achieved with an acidic BGE were much narrower than with a basic one. The results implied that the theoretical sample injection length with the former should be much longer than with the latter, which has been verified by a number of subsequent reports. For instance, under acidic sweeping-MEKC conditions, the injection length was 41.16 cm for linezolid [25], 10.9 cm for neonicotinoid insecticides [26] and 15 cm for steroids [27]; however, for alkaline sweeping-MEKC, 5.52 cm for all transand 13 cis-retinoic acids [28], 4.66 cm for neutral steroids [29] and 2.45 cm for aflatoxins [30]. In this study, both acidic and basic BGEs were investigated and optimized, and the results obtained were compared. In preliminary experiments, the baseline separation could not be achieved in an acidic BGE with an amount of CH3 CN < 20% v/v. Different contents of CH3 CN (25– 35%) were initially investigated in conjunction with the buffer containing 25 mM NaH2 PO4 and 50 mM SDS (pH 2.3 adjusted with 3 M H3 PO4 ). And the results showed that 30% CH3 CN was optimal. Subsequently, the concentrations of the NaH2 PO4 buffer (10–30 mM) and SDS (30–60 mM) were ex C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Effect of -CD concentration in BGE. Experimental conditions: BGE, 55 mM SDS + 10% v/v CH3 CN + 25 mM borate buffer (pH 9.2); other conditions: 28 kV; sample matrix, 30 mM borate buffer (pH 9.2, containing 10% v/v CH3 OH); injection length, 17.4 cm. Concentration of each PAE: 0.5 g/mL; Peaks: 1, DEP; 2, DIBP; 3, DBP; 4, BBP.
amined, and 10 mM NaH2 PO4 and 40 mM SDS were adopted. To improve the resolution between BBP and DBP, -CD (0–15 mM) was added into the buffer due to its powerful separation capacity for lipophilic compounds with similar structures, but the results showed no improvement was achieved. It could be speculated that CH3 CN was functioning as a competitive substance; as its concentration was increased, to a certain extent it effectively competed to displace the target analytes from the guest cavity of -CD [31], which restrained the capacity of -CD partitioning PAEs. The NaH2 PO4 content varying from 10 to 30 mM in the sample solution containing 10% v/v CH3 OH was investigated, and the result indicated it had no positive influence on the focusing effect and resolution. Sample zone length injected ranging from 8.7 to 34.8 cm was examined, and the results demonstrated broaden bands and poor resolution occurred with the length exceeding 18 cm. Eventually, a sample matrix of 10 mM NaH2 PO4 and an injection length of 17.4 cm were used. The sweeping--CD-MEKC method under alkaline BGE was studied. Firstly, the -CD concentration (0–10 mM) was modified with the buffer consisting of 25 mM borate buffer and 55 mM SDS (pH 9.2) containing 10% v/v CH3 CN. As shown in Fig. 1, both resolution and sensitivity were poor for DIBP, DBP and BBP without incorporation of -CD, but 5 mM -CD gave good resolutions, which was used in subsequent investigations. The concentration of borate buffer concentration (15– 30 mM) and the pH value (8.5–10.0) were tested. The complete separation was obtained at 25 mM borate with a pH value of 9.2. The SDS concentrations (50–80 mM) were also studied, and results showed that 50 mM SDS was the optimal value. 9% v/v CH3 CN was used to further improve the separation. www.jss-journal.com
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A
Figure 2. Effect of borate concentration in sample matrix. Experimental conditions: BGE, 5 mM -CD + 50 mM SDS + 9% v/v CH3 CN + 25 mM borate buffer (pH 9.2); Concentration of each PAE: 2.0 g/mL; other conditions: 22 kV; injection length, 13 cm.
The effect of the conductivity in the sample matrix on the focus effect was examined by dissolving the analytes (2.0 g/mL) in the buffers containing 30–80 mM borate (pH 9.2) with 10% v/v CH3 OH. As shown in Fig. 2, when increasing the borate content from 30 to 80 mM, the focus effect was improved and the peak shapes tended to be better. A sample matrix with higher conductivity (e.g. 80 mM borate buffer) produced a reduced electric field in sample zone and an enhanced electric field in BGE region. When voltage was applied, SDS stacked in the front of high conductivity sample zone. In this case, the concentration of SDS entering the sample zone was higher than under a homogenous electric field or an enhanced electric field in the sample zone. Hence, the analytes were picked up and accumulated by the stacked SDS penetrating the sample zone, and the final swept zone was narrower. Therefore, 80 mM borate buffer was selected. Sample injection length was investigated from 8.7 to 26.1 cm. The results demonstrated that increasing the injection length decreased the migration time, and the resolutions and peak shapes were gradually deteriorated with the length of injected sample > 13 cm. As a result, a sample plug of 13 cm was chosen in this study. Figure 3 showed typical CE electropherograms of PAEs standard obtained in the acidic (Fig. 3A) and alkaline buffers (Fig. 3B), respectively. As illustrated, although slightly better enrichment effect was achieved with the acidic BGE, the basic BGE provided a much shorter migration time and baseline resolution, which was eventually selected.
3.2 Optimization of DLLME conditions In the previously reported DLLME procedures for the analysis of PAEs in water samples, the extractant mixture used were CCl4 /CH3 CN [32] and C6 H5 Cl/acetone [33]. In this study, as C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B
Figure 3. Typical electropherograms of PAEs standard (1 g/mL) by the acidic (A) and alkaline separation conditions (B). Experimental conditions: (A), BGE, 40 mM SDS +30% v/v CH3 CN + 10 mM NaH2 PO4 (pH 2.3); other conditions, –22 kV; injection length, 17.4 cm. (B), BGE was the same as Fig. 2. Peaks of Fig. 3A: 1, BBP; 2, DBP; 3, DIBP; 4, DEP.
Chinese white spirit samples usually contain about 50% v/v ethanol, which can dissolve most of DLLME extractants, no additional disperser was therefore required. In this context, the effects of various experimental parameters, such as the type and volume of extraction solvents, the disperser volume and salt addition, on DLLME efficiency were optimized. In the preliminary experiment, it was found that if adopting 20% v/v ethanol as the disperser, there was scarcely any sedimented phase after microextraction and centrifugation. Therefore, 2 mL Chinese white spirit sample mixed with 8 mL 10% v/v NaCl solution was used to optimize the parameters. 120 L of different extraction solvents including CCl4 (1.59 g/mL), CHCl3 (1.47 g/mL), CH2 Cl2 (1.32 g/mL) and C6 H5 Cl (1.10 g/mL) were tested. When using CH2 Cl2 , the sedimented phase could not form, which could be due to its comparatively high solubility in water (the solubility of CH2 Cl2 , CHCl3 , CCl4 and C6 H5 Cl at 20⬚C in water are 20, 8, 0.8 and 0.49 g/L, respectively). C6 H5 Cl and CHCl3 provided low extraction recoveries% (ERs%) for DBP and www.jss-journal.com
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Table 1. Linearity, calibration curve, r, LODs, LOQs, EF and repeatability data obtained for a spiked Chinese white spirit sample
Linearity (ng/mL) Calibration graphs Calibration curve (n = 7) r LODs (ng/mL) LOQs (ng/mL) Enhancement factor (EF) DLLME Sweeping--CD-MEKC DLLME-sweeping--CD-MEKC Reproducibility (RSD%) Peak area 20 ng/mL 100 ng/mL 300 ng/mL Migration time 20 ng/mL 100 ng/mL 300 ng/mL
DEP
DIBP
DBP
BBP
10500
51000
51000
5800
Y = 101.83X + 1070.2 0.9909 0.8 2.7
Y = 98.96X + 828.58 0.9988 0.6 1.9
Y = 86.45X + 722.26 0.9969 0.5 1.6
Y = 104.59X + 618.17 0.9990 0.4 1.4
4 25 92
3 25 81
3 24 79
3 26 82
5.7 3.5 2.9
3.7 4.8 3.7
5.9 4.3 2.5
6.7 4.8 4.9
2.1 1.6 1.2
1.6 1.3 2.8
2.1 1.7 1.9
1.9 2.3 2.0
DIBP. Among these extraction solvents, CCl4 gave the highest ERs% for all the target analytes, thus it was selected for subsequent experiments. To illustrate the effect of the disperser volume on ERs%, the different volumes of Chinese white spirit (1–4 mL) were diluted to the bulk volume of 10 mL with aqueous NaCl solution, which produced the sample matrices containing 5, 10, 15, and 20% v/v ethanol, respectively. The results showed that the best ERs% of PAEs was obtained when using 2 mL Chinese white spirit sample. The volume of CCl4 was tested in the range of 60– 140 L in 20 L intervals. It was observed that the ERs% rapidly increased from 60 to 100 L, after which it remained constant until the CCl4 volume was increased up to 140 L. On the basis of this result, 100 L CCl4 was utilized for the following experiments. The different concentrations of NaCl ranging from 0 to 20% w/v were evaluated. The results showed that, increasing the salt content from 0 to 10% w/v, the ERs% of each analyte increased gradually; with the amount of NaCl exceeding 10% w/v, the ERs% varied negligibly. It could be explained that ERs% increased due to salting out, whereby water molecules form hydration spheres around the ionic salt molecules that reduce the concentration of water available to dissolve the analytes molecules, thereby driving the additional analytes into the organic droplets [19]. Therefore, 10% w/v NaCl was used. In summary, the optimal extraction procedure was performed by adding a mixture of 100 L CCl4 and 2 mL Chinese white spirit to 8 mL salt solution (containing 1 g NaCl), followed by a manual shaking time of 3 min and centrifugation time of 3 min at 4000 rpm. The sedimented phase was evaporated at 30⬚C under nitrogen stream until dryness and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
re-suspended with 0.5 mL 80 mM borax containing 10% v/v CH3 OH.
3.3 Method validation and comparison with other analytical procedures Under optimum conditions, method performance was evaluated in terms of linearity, LODs, LOQs, enhancement factors (EFs) and reproducibility. Good linearity was observed for all the analytes, and the regression equations were shown in Table 1. The LODs (S/N = 3) ranged from 0.4 to 0.8 ng/mL, and the LOQs (S/N = 10) were between 1.4 and 2.7 ng/mL. The EFs with DLLME-sweeping--CD-MEKC as compared to conventional MEKC were in the range of 79- to 92-fold. As can be seen in Table 1, the results showed acceptable reproducibility in all cases with RSD% values for peak area and migration time less than 6.7 and 2.8%, respectively. As shown in Table 2, the recoveries of the four PAEs were in the range of 89.6–105.7% with RSDs% less than 6.3%. Table 3 showed the comparison of the proposed approach and other reported methods by virtue of organic solvent consumption, extraction time, LOQs and EFs. The volume of organic solvent consumed in this method (100 L CCl4 ) was much less than the SPE-MEKC (29 mL CH3 OH) [12]; the LOQs obtained in this study (1.4–2.7 ng/mL) were lower than the LOQs of the IL-DLLME (10.6–28.5 ng/mL) [18], sweeping-MEEKC (75–150 ng/mL) [34] and SPE-MEKC (21400–57700 ng/mL) [12]. Although the USVA-DLLME–GC with flame ionization detection (FID) and ion trap mass spectrometry (ITMS) procedure consumed less extraction solvent (40 L CH2 Cl2 ) [4] and provided lower LOQs www.jss-journal.com
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Table 2. Spiked recoveries of PAEs in real samples by the proposed method (n = 3)
Samples
Added (ng/mL)
DEP Recovery% (RSD%)
DIBP Recovery% (RSD%)
DBP Recovery% (RSD%)
BBP Recovery% (RSD%)
S-2
50 150 300 20 150 300 50 150 300
92.8 (2.6) 96.4 (2.4) 94.7 (3.1) 92.7 (2.6) 94.6 (4.2) 92.1 (2.2) 92.7 (2.6) 95.3 (2.4) 95.9 (3.0)
92.3 (4.7) 93.7 (1.2) 91.9 (3.4) 90.5 (2.0) 105.7 (5.5) 91.4 (3.1) 92.4 (3.9) 94.1 (2.8) 90.4 (1.2)
91.5 (4.7) 92.6 (6.3) 89.8 (3.5) 95.3 (1.9) 93.1 (2.9) 90.7 (1.4) 93.5 (4.0) 95.2 (2.3) 92.3 (1.7)
90.9 (1.2) 94.6 (5.3) 91.6 (3.1) 90.4 (1.3) 94.9 (2.9) 89.6 (3.8) 91.2 (5.6) 94.5 (4.2) 91.3 (3.2)
S-5
S-10
Table 3. Comparison of the current work with other previously reported methods for the determination of PAEs
Method
Sample volume Matrix
Organic solvent consumption
Extraction time (min)
LOQs (ng/mL)
RSD%
Enrichment factor
Ref
10.6–28.5
7.8–15
—
[18]
0.4–1.3
1.5–3.4
14-49
[17]
0.08–0.4
4.9–8.2
220-300
[4]
IL-DLLME–HPLC–UV a) 10 mL
Water
MSA-DLLME–HPLC– UV b) USVA-DLLME–GC–FID/ ITMS c) Sweeping-MEEKC– DAD SPE–MEKC–DAD DLLME-sweeping-CD-MEKC–DAD
10 mL
Drinking+water
10 mL
Wine
Acetone (1 mL) 15 + [C8 MIM][PF6 ] (80 L) Dodecane 10 (200 L) CH2 Cl2 (40 L) 15
—
Soft drinks
—
—
75–150
4.9
—
[34]
500 L 2 mL
Perfume Chinese white spirit
CH3 OH (29 mL) CCl4 (100 L)
20 15
21,400–57,700 1.4–2.7
2.5–11.6 2.5–6.7
— 79–92
[12] This method
a) Ionic liquid-based-DLLME–HPLC–UV. b) Magnetic stirring-assisted-DLLME–HPLC–UV. c) Ultrasound-vortex-assisted-DLLME–GC–FID/ITMS.
(0.08–0.4 ng/mL) than the current method, the preliminary experiment showed CH2 Cl2 could not present efficient extraction for PAEs in Chinese white spirits.
3.4 Assays of real Chinese white spirit samples The proposed DLLME-sweeping--CD-MEKC method was applied to determine the four PAEs in 22 Chinese white spirits. The typical electropherograms for S-5 (a) and spiked S-5 (b) and S-12 (c) are depicted in Fig. 4, and the assay data are shown in Fig. 5. As illustrated in Fig. 5, among the 22 Chinese white spirits, DEP and BBP were found in low levels and frequencies, but the detection frequencies for DIBP and DBP were 95.5 and 100%, respectively, which were also frequently found in previous works [2–4, 15]. The contents of DBP in 55% of the Chinese white spirit samples exceeded the established C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SML (0.3 mg/kg) in Directive 2007/19/EC, and the maximum (5.569 g/mL) was about 18.6 times with respect to the SML. Although it is difficult to give information about the source of DIBP or DBP in Chinese white spirit on the basis of the experimental results, it is worth noting that people should refrain from over-drinking Chinese white spirit and pay more attention to its possible PAEs contamination.
4 Conclusions A novel method based on DLLME coupled with sweeping-CD-MEKC was successfully developed for the determination of PAEs in Chinese white spirits. The presented method accomplished the baseline resolution among DIBP, DBP and BBP within 13 min, exhibiting significantly higher separation efficiency as compare with HPLC procedures. The combination of DLLME and sweeping-MEKC enabled the www.jss-journal.com
Electrodriven Separations
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quantitative analysis of PAEs at ng/mL level, which was much lower than the SML (0.3 mg/kg) established by the European Union and the Chinese national standard. Moreover, the present procedure offered the advantages of simplicity, low consumption of toxic organic solvents and low cost. Equipped with the merits and features, this method could be exploited as an alternative routine analysis method for the PAEs analysis in aqueous matrix. The authors acknowledge with gratitude and appreciation financial support from Northwest A&F University. The authors have declared no conflict of interest. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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