Food Science and Technology Research, 24 (2), 231_239, 2018Copyright © 2018, Japanese Society for Food Science and Technologydoi: 10.3136/fstr.24.231

http://www.jsfst.or.jp

*To whom correspondence should be addressed. E-mail: lihuabin@mail.sysu.edu.cn

Original paper

Optimization and Application of Ultrasound Assisted QuEChERS and Ionic Liquid Dispersive Liquid–liquid Microextraction Followed by HPLC for Determination of BBP and DBP in Packaging Food

En-Qin Xia1,2†, Yonghong Chen

3†, Qin Lu3, Ya Li

1, Ying hang2, Jiewen Su

2, Yuting Liu2 and Hua-Bin Li

1*

1Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China

2Dongguan Key Laboratory of Environmental Medicine, School of Public Health, Guangdong Medical University, 523808 Dongguan, China

3Guangdong International Travel Healthcare Center, Guangzhou, 510635, China

Received September 11, 2017 ; Accepted November 28, 2017

A simple, rapid and efficient method named ultrasound-assisted QuEChERS (quick, easy, cheap, effective, rugged, and safe) and ionic liquid dispersive liquid-liquid microextraction (IL-DLLME) prior to high performance liquid chromatography with photodiode array detection (HPLC-PAD) was developed for the determination of butyl benzyl phthalate (BBP) and di-n-butyl phthalate (DBP) from packaged fowl meat products. The optimum conditions for IL-DLLME experiments were tracked by response surface methodology (RSM). Under the optimum conditions, the precision of proposed method less than RSD 4.0% and linear working range of 5 to 500 μg L

_1 with R2 above 0.9985 were achieved. The LOD was verified as 0.25 and 0.52 μg L

_1 for BBP and DBP, respectively. The mean recoveries ranged from 86% to 98% with RSD less than 10.1%. The proposed method was successfully detected the presence of BBP, DBP in canned and plastic food. No significant increase of migration of the two analytes from packages to food contacted during 9 months of shelf life was detected.

Keywords: butyl benzyl phthalate, dibutyl phthalate, ionic liquid dispersive liquid-liquid microextraction, high performance liquid chromatography

IntroductionNowadays, packaging is applied on a huge variety of

products to protect food during all stages of the supply chain. Phthalates (PAEs), a group chemicals of dialkyl or alkyl/aryl esters of phthalic acid, are the most common plasticizers in plastic packaging, which may amount to 20%‒40% (w/w) in plastic products. As low molecular weight compounds and not bound chemically in the plastics, PAEs can be readily dispersed into the foodstuff from food plastic containers during the processes of production, use, or disposal (Pedersen et al., 2008). PAEs are considered as endocrine disrupter

chemicals (EDCs) because they affect reproduction and development of organisms by disturbing hormone synthesis in animal studies, which suggest that they are potential toxicants for human health (Biscardi et al., 2003; Gray et al., 2000). In commercial personal care products (shower gels), phthalates were simultaneously detected by a simple dilution, and the combining effect on health was proved even greater (Esteve et al., 2016). The public concern on their migration into food greatly increases, especially for food with longer shelf life. In the PAEs family, di-(2-ethylhexyl) phthalate (DEHP), butylbenzyl phthalate (BBP) and dibutyl phthalate (DBP) are

E.-Q. Xia et al.232

major members in terms of their worldwide application and potential effects. BBP and DBP have been limited at 30.0 mg kg

_1 and 0.3 mg kg_1 of the migration levels for food simulants

by Commission of the European Communities in 2007, respectively. DBP has also been limited at the level of 0.3 mg kg

_1 in food in China (Fasano et al., 2012, Fasano et al., 2015). Moreover, according to the results of Fierens’ work group, BBP has been found several hundred higher levels than DEHP in packaged poultry meat samples. DBP concentrations were the highest in food packaging materials (Fierens et al., 2012). Therefore, BBP and DBP may be the main responsible members involving in the migration event for packaged fowl meat products in the PAEs family. Development of the analytical methods for monitoring the presence and migration of DBP and BBP into large amount of packaging fowl meat products are particularly urgent to regulate the vigorous development of food industry.

Several techniques have been used for determination of PAEs, mainly including gas chromatography (GC) and high-performance liquid chromatography (HPLC) equipped with various detectors (Du et al., 2016; Gallart-Ayala et al., 2013; Gu et al., 2014; Liu et al., 2015; Wang et al., 2015) .For detecting trace levels analytes in complex food matrices, a cleanup/preconcentration step is necessary before instrumental analysis. Various sample pretreatment methods such as liquid liquid extraction (LLE), solid phase extraction (SPE), headspace solid-phase microextraction, matrix solid phase dispersion, pressurised liquid extraction (PLE) have been developed (Liu et al., 2015; del Carlo et al., 2008; Feng et al., 2016; Capriotti et al., 2015). However, some disadvantages of them, such as large organic solvents comsumption for LLE, high requirements in cost, speciality and skill for SPE and high energy consumption for PLE, prevent them from wide application. Recently, ionic liquids (ILs) (e.g. alkyl methylimidazolium hexafluorophosphate) were tested as extraction solvents in DLLME (dispersive liquid-liquid microextraction), which is a modified DLLME method as IL-DLLME method with several advantages, such as less toxicology, rapidity, simplicity of operation, low cost (Rezaee et al., 2010; Abdelhamid 2016). In addition, the characteristic of ILs, i.e., tunable viscosity by temperature and miscibility with water and organic solvents, markly improves the application of DLLME method. However, application of IL-DLLME method to pretreatment solid food samples is a challenging work for its limited selectivity. Efficient and very simple sample treatments based on QuEChERS (quick, easy, cheap, effective, rugged, and safe) methodology have been optimized for each type of matrix (Huertas-Pérez et al., 2016). Samples extraction and phase separation according to the modified QuEChERS method before IL-DLLME might be perfect to deal with solid food complex matrixes. Moreover, sonication

can accelerate the mass-transfer process between two immiscible phases during extraction, which led to a rapid increment in the extraction efficiency in a minimum amount of solvent and time (Xia et al., 2011; Xia et al., 2012; Kuo et al., 2013). Ultrasound radiation conducted during QuEChERS extraction and IL-DLLME could be an attractive and economical tool for extraction of BBP and DBP from complicated food samples. Response surface methodology (RSM) is an attractive collection of statistical techniques for developing and optimizing complex extraction processes. According to RSM, evaluation the effect of independent variables and their interaction effects could be achieved at the same time by decreasing work and number of trials compared to single-factor experiment (Arvindekar et al., 2016; Zhao et al., 2007).

In this study, the purpose was to set up a simple, low cost, rapid and low organic solvent-consuming analytical method for the simultaneous determination of two PAEs by ultrasound-assisted extraction (UAE) combining with UA-IL-DLLME before HPLC- photodiode array detector (PAD). The main parameters affecting the efficiency of UA-IL-DLLME were optimized by RSM. Besides, the optimized method was tried to monitor BBP and DBP migrated from cans and bags into food during shelf life.

Materials and Methods

Reagents HPLC-grade solvents (acetonitrile, methanol, acetone and ethanol) were purchased from Merck Chemical Company (Darmstadt, Germany). The ionic liquids, i.e., 1-butyl-3-methylimidazolium hexaflorophosphate [C4MIM][PF6], 1-hexyl-3-methylimidazolium hexaflorophosphate [C6MIM][PF6], 1-octyl-3-methylimidazolium hexaflorophosphate [C8MIM][PF6) were obtained from Taide Chemical Company (Guangzhou, China). BBP and DBP were purchased from Sigma-Aldrich Laborchemikalien GmbH (Seelze, Germany). The distilled water was used throughout the experiment.

All glass wares employed were baked over 350℃ for 4h in muffle after a rigorous washing with organic solvents to prevent phthalate contamination.

Individual stock standard solutions (100 mg L_1) of

corresponding analytes were prepared in methanol. The mixed standard solutions for HPLC analysis were prepared by diluting the stock solutions with methanol. All standard solutions were stored at 4℃. The working standards of analytes were prepared from the suitable dilution of stock standard solution with methanol on site.

Apparatus An ultrasonic cleaner (KQ600E, Kunshan Ultrasonic Instrument, Jiangsu, China) was used for acetonitrile extraction and emulsification of the solutions. A Hettich Universal 320 R centrifuge was used to accomplish DLLME procedure.

The HPLC-UV analysis was performed on a Waters (Milford, MA, USA) 1525 Binary pump separations module, equipped with

UA-QuEChERS-ILDLLME-HPLC for Determination of BBP and DBP in Packaging Food 233

a Waters 2996 photodiode array detector. Evaluation and quantification were performed with an Empower Chromatography System (Waters, America). The analytical system also included an auto-injector, and a Waters reversed-phase C18 analytical column (Agilent, 4.6 mm × 250 mm, 5 μm). Isocratic elution was used and the mobile phase was acetonitrile-water (75:25, v/v) with a flow-rate of 0.8 mL min

_1 at room temperature. The UV spectra were recorded between 190 and 400 nm for peak characterization, and the detection wavelength was set at 280 nm. The retention time of BBP and DBP were 12.33 and 14.19 min, respectively (Fig. 1).

Sample treatment Food samples were purchased from markets in Guangzhou, Guangdong Province, China. The unopened packages were stored at room temperature. After the packages were opened, the total solid contents of each package were immediately homogenized and stored in vinyl bags at ‒20℃ until analysis. The fowl meat samples such as canned chicken in pepper oil, plastic packaged chicken in sauce, canned duck meat and plastic packaged duck meat were tested.

Ultrasound assisted QuEChERS Based on QuEChERS method, 5 g of thoroughly homogenized solid sample was transferred into a 20 mL screw-capped glass vial with 5 mL of deionized water. The vial was sealed and vortex-mixed for 4 min. Then, 10 mL of acetonitrile, 4 g of anhydrous MgSO4 and 1 g of NaCl were added, and the vial was sealed and placed at ultrasonic cleaning tank for 20 min. The mixed solution was centrifuged for 5 min at 3500 g and the residue was washed twice with 5 mL acetonitrile. The combined supernatants were defatted twice with 10 mL hexane for 5 min. The acetonitrile layer was collected and evaporated near to dryness by rotary evaporation at 35℃. The residue was dissolved with 311 μL of dispersive solvent and stored at 4℃ until UA-IL-DLLME procedure.

Ionic liquid dispersive liquid−liquid microextraction procedures (IL-DLLME) A homogeneous mixture of 311 μL of extract solution or standard solutions and 59.7 μL (C6MIM) (PF6) (as extraction solvent) was rapidly injected into 10-mL glass centrifuge tubes with a conical bottom containing 5 mL of water by 1 mL of syringe. A cloudy solution was immediately formed by the dispersion of fine droplets of the ionic liquid throughout the sample solution. The tube was sealed and placed in the ultrasonic cleaning tank. The cloudy condition was kept for 22.95 min and at 30℃ by

ultrasound radiation. The solution was then centrifuged for 5 min at 3500 g. The lower IL phase was collected and dilution with 50 μL acetonitrile to reduce the viscosity of the IL, and then introduced into HPLC system for analyses.

Experiment design Method optimization mainly involved the maximization of DLLME recovery of a standard solution with two target analytes as a function of six independent variables, namely, extraction solvent volume, disperser solvent volume, pH, amount of NaCl, sonication temperature and time. The types of extraction and dispersive solvents are also known to affect the extraction efficiency. For selecting the best solvent, the level of the factors above were fixed, and the standard solutions at pH 6 was extracted by 50 μL of extraction solvent and 500 μL of dispersive solvent at room temperature for 15 min. The optimal dispersive solvent and extraction solvent were chosen according to the mean recovery of target analytes. Then, the multilevel single factorial experiments were conducted to achieve the influential parameters affecting the IL-DLLME efficiency, namely, the volume of dispersive and extraction solvents (acetonitrile and IL, respectively), the sonication time. Their varying ranges and center point for next multilevel central composite design (CCD) were obtained. The suitable experimental conditions for pH, ionic strength, extraction temperature of the IL-DLLME solution were also tracked.

The Design-Expert Software (Version 7.1.3, Stat-Ease, Inc., Minneapolis, USA) was employed to complete the CCD design and the statistical analysis of data. An optimization design of twenty experiments with 6 center points was selected. The set conditions of two significant factors and the responses are listed in Table 1. Then, the quadratic polynomial model Eq. (1) was used to evaluate the functional relationship between each of the responses and the set of independent variables, and the effect of the independent variables interaction on responses as well. A suitable approximation of the response and the variable conditions were predicted, simultaneously. P value of less than 0.05 was considered to be statistically significant. All the tests were conducted in triplicate and averaged.

yk = bk0 + Σ3

i = 1 bkixi + Σ

3

i = 1 bkiixi + Σ

3

i = 1 bkilxixj ······Eq. 1

Where, yk was expressed as the response function, the coeffcients of the polynomial, bk0, bki (linear effects), bkii (quadratic effects), and bkij (interaction effects), represented the constant regression coefficients of the model, and xi and xj were the independent variables (Table 1).

Results and DiscussionIL-DLLME optimizationThe multilevel single factorial optimization From the

multilevel single factorial experiments, (C6MIM)(PF6) and acetonitrile were chosen as extraction and dispersive solvent due to their releasing higher extraction efficiency for the two analytes than other solvents tested (i.e. [C4MIM][PF6],

Fig. 1. The chromatograms of standard solution of DBP and BBP.

E.-Q. Xia et al.234

[C8MIM][PF6], methanol, acetone and ethanol). The suitable level three factors, including pH, amount of NaCl, sonication temperature were achieved, and the results were showed in Fig. 2. Usually, aqueous samples pH influence the existing forms of the analytes, which impact the extraction efficiencies of targets. Different pH values in the range of 5.0‒10.7 were tested, as illustrated in Fig. 2c. The results showed that the extraction efficiencies were nearly peak at pH 6.2 for DBP and BBP, which indicated that DBP and BBP as esters are even unstable in non-acidic medium. For organic extraction solvent, the addition of salts (such as NaCl) in aqueous samples can decrease the solubility of analytes and enhance their partitioning into the organic phase (salting-out effect), which sometimes improves recovery (Wang et al., 2015;Zhao et al., 2007). However, in this work, the extraction recoveries of analytes were almost constant in the presence of NaCl in range of 0‒25% (w/v). The results are shown in Fig. 2d. Effect of aqueous solution temperature (20‒60℃) on the extraction efficiencies of targets were tested, as illustrated in Fig. 2e. The extraction efficiencies were nearly maximum at range of 30‒40℃. Therefore, no addition of NaCl, pH 6.2, and temperature of 30℃ were chosen as fixed parameters for the two analytes in the CCD optimization.

The other three factors, including sonication time (A), the volume of acetonitrile and IL (B, C), show significant effect on the extraction efficiencies in the ranges of 5‒40 min, 100‒1000 μL and 20‒80 μL, respectively (Fig. 2a, b, f). The peak recovery of DLLME was obtained at 50 and 400 μL of the IL and acetonitrile,

respectively. Sonication time showed maximum effect at 20 min. The optimal data would be implemented as the center points for the further CCD procedures. Therefore, their three fixing levels (‒1, 0 1) for CCD were also obtained and showed in Table 1, i.e., extraction time (15, 20, 25 min), the volume of dispersive solvents (300, 400, 500 μL), and the volume of extraction solvents (40, 50, 60 μL).

The multilevel central composite optimization From the design and Eq.(1), the quadratic polynomial models for average recovery of the two analytes was achieved Eq. (2) as following:

R=91.23+2.14A+2.61B‒0.53C+0.14AB‒0.24AC‒3.05BC‒2 .49A2‒0.76B2‒2.10C2 ······Eq. 2

Where, R is the predicted response value, i.e., DLLME recovery percentage, and A, B and C are the coded values of variables, including sonication time, the volume of disperser solvent (acetonitrile) and the volume of the extraction solvent volume (IL), respectively.

An analysis of variance (ANOVA) about the response was conducted according to the Design Experts Software (Table 2). The values of ‘Prob > F’ less than 0.05 with an F value of 4.67 indicated that model terms were significant. The interaction of the volume of acetonitrile and the volume of IL (BC) were found to be significant (0.01 < p < 0.1). However, no significanct interaction between sonication time and other variables (AB or AC) was found (p > 0.1). The non-significant lack of fit (p > 0.05) conformed the validity of the model was adequate for the response.

From Eq. (2), the positive coefficients of variables

Table 1 . Matrix of the central composite experimental design and mean responses.

Run PatternSonication time Acetonitrile IL Mean response

(%)(A, min) (B, μL) (C, μL)

1 (1,1,‒1) 25 500 40 96 .782 (0,0,0) 20 400 50 91 .613 (‒1,‒1,‒1) 15 300 40 75 .694 (0,0,1 .68) 20 400 66 .8 84 .285 (‒1,‒1,1) 15 300 60 81 .666 (0,0,0) 20 400 50 86 .857 (1,‒1,1) 25 300 60 86 .818 (0,0,0) 20 400 50 92 .559 (1,‒1,‒1) 25 300 40 82 .1910 (0,1 .68,0) 20 568 50 89 .8511 (1 .68,0,0) 28 .4 400 50 85 .812 (1,1,1) 25 500 60 89 .5613 (‒1,1,1) 15 500 60 83 .4714 (0,0,0) 20 400 50 91 .7815 0,1 .68,0 20 232 50 88 .6516 ‒1,1,‒1 15 500 40 90 .0917 (0,0,0) 20 400 50 92 .1318 (0,0,0) 20 400 50 92 .4119 (‒1 .68,0,0) 11 .6 400 50 82 .9420 (0,0,‒168) 20 400 33 .2 86 .66

UA-QuEChERS-ILDLLME-HPLC for Determination of BBP and DBP in Packaging Food 235

Table 2 . An analysis of variance (ANOVA) about the response.

Source Sum of Squares Degrees of Freedom Mean Square F Value P Value

Model 374 .6987402 9 41 .63319 4 .677456 0 .0122 significantA-A 62 .6467838 1 62 .64678 7 .038316 0 .0242B-B 92 .6891521 1 92 .68915 10 .41355 0 .0091C-C 3 .86165475 1 3 .861655 0 .433854 0 .5250AB 0 .158058364 1 0 .158058 0 .017758 0 .8966AC 0 .481180101 1 0 .48118 0 .05406 0 .8208BC 74 .56874404 1 74 .56874 8 .377738 0 .0160A^2 88 .81544552 1 88 .81545 9 .978344 0 .0102B^2 8 .252743294 1 8 .252743 0 .927189 0 .3583C^2 63 .08991991 1 63 .08992 7 .088102 0 .0238Residual 89 .00820495 10 8 .90082Lack of Fit 65 .40593566 5 13 .08119 2 .771171 0 .1438 not significant

Fig. 2. Effect of the multilevel single factors on the DLLME: a, the volume of extract solvent (ILs); b, the volume of disperse solvent (acetonitrile); c, pH; d, the concentration of NaCl (%); e, extraction temperature; f, extraction time.

E.-Q. Xia et al.236

indicated that an increase in sonication time (A) and the volume of acetonitrile (B) could bring about positive extraction efficiency, while it is contrary for the volume of IL (C) with the negative coefficient. From ANOVA evaluation, the sonication time (A) and the volume of acetonitrile (B) were highly significant at p < 0.05. The influence of the volume of acetonitrile on the extraction yield was even higher. The sonication time (A) did not show significant effects (p > 0.1) on even extraction efficiency of analytes.

According to Eq. 2, the optimum level of each variable, and effects of their interaction on IL-DLLME were studied by plotting three-dimensional response surfaces and two-dimensional contour lines (Fig. 3). The plots easily revealed visually effect and interactions between two independent variables on the response at the experimental range, with the third variable kept constant at its optimum level. The contour plots showed the influential interaction between the volume of acetonitrile (B) and that of IL (C) in Fig. 3c, while the interactions of two variables displayed in Fig. 3a and 3b were insignificant. From three-dimensional response surfaces, the higher extraction efficiency occurred at the high level of IL volume (C) and at the medium level of acetonitrile (B) volume, which indicated that IL was fine efficiently dispersed in the aqueous solution, and then restored to form setting phase under the conditions. However, no significant effect of sonication time (A) on the response value was found.

The finally appropriate variable conditions with a highly desirable value were predicted by software as sonication time, volume of acetonitrile and IL were 22.59 min, 311 μL and 59.6 μL, respectively.

Under the optimal conditions, the model predicted recovery of two average analytes was 98.10%. Then the verification tests with the optimal conditions were conducted three times. The average recovery of the two analytes was 97.69% with 3.5% of the RSD, which confirmed the sufficiency and accuracy of the response optimization model.

Method validation The proposed method was evaluated in term of the linearity, limits of detection (LODs), limits of quantification (LOQs), inter- and intra-day repeatability and recovery of each compounds investigated. The cross contamination from chemicals, materials and laboratory equipment was strictly checked and controlled for BBP and DBP determination.

The l ineari ty , l imits of detect ion (LODs), l imits of quantification (LOQs), precision and recovery of the proposed method The linearity of calibration curve was checked by preparing standard solutions of each analytes at seven different concentrations using the stock solution. The calibration curves of BBP and DBP concentrations were constructed ranging of 5 to 500 μg L

_1and 10 to 500 μg L_1, respectively, with correlation

coefficients (R2) more than 0.9985 (Table 3).According to literature, the values of the LODs and LOQs

are defined based on the concentration of an analyte that produces a chromatographic peak equals to 3-times (LOD) and 10-times (LOQ) the standard deviation of the baseline noise (a signal/noise of 3:1 and 10:1, respectively) (Xia et al., 2011). From Table 3, the LODs (S/N=3) ranged between 0.25 and 0.52 μg L

_1 and the LOQs (S/N=10) ranged between 0.83 and 1.75 μg L

_1 for two analytes. The same samples were repeatedly analyzed 7 times over

two days for precision about each analytes at the concentration of 10 μg L

_1 and 100 μg L_1. The relative standard deviation

Fig. 3. Three dimensional plots of the interacting effect of the independent variables on UA-IL-DLLME recovery: a extraction (IL) versus sonication time, b volume of dispersive solvent versus sonication time, c volume of extractor solvent versus volume of dispersive solvent (The red marks in a and b mean design points above the predicted values).

UA-QuEChERS-ILDLLME-HPLC for Determination of BBP and DBP in Packaging Food 237

(RSD) values of the analyses conducted in the same day and two consecutive days were below 4% in all cases (Table 3). The results indicated good instrument reproducibility and chromatographic behavior of all the compounds investigated.

The recovery test was conducted by spiking at 2 concentration levels (2 and 10 μg kg−1) of the two analytes to the homogenized samples to evaluate the accuracy of the proposed method, and the results are also listed in Table 3. The results showed the recoveries ranged between 87%‒96% and 86%‒98% for canned chicken in pepper oil and plastic packaged chicken in sauce, respectively, with standard deviations (SD) ranging from 4.4% to 10.1% in all the cases. Furthermore, relatively good mean recoveries were showed at the 2 μg kg

_1 addition for the two analytes. The results demonstrated the high stability and applicability of the proposed method for the determination of the two analytes, even the analytes concentration near to LOQ.

Comparison of LOD of the proposed method with other methods LOD is known as the most important parameters in

quantitative analysis (Sengul 2016). In the literature, the PAEs were frequently detected by GC or LC system coupled with mass spectrometry. The pretreatment procedure usually involves SPE, which is also widely applicated method for excellent selectivity with higher requirements in cost, speciality and skill than that of IL-DLLME in this work. Table 4 shows comparison of the LOD determined in this study with those reported in literatures. Compared to the previous methods, the proposed method displayed similar or lower LOD, even to that of the previously reported methods using mass spectrometer followed SPE. The proposed method might be a powerful method for screening DBP and BBP in complex food solid samples due to its significant advantages, such as easily operating, low cost, less time and less organic solvents consuming.

Analysis of real samples The established method was successfully applied for the analysis of DBP and BBP in different commercial food samples at different production date, such as plastic packaged chicken in sauce, canned chicken in pepper oil,

Table 3 . The Retention time, Linear Range, Limit of Detection (LOD), Limit of Quantitation (LOQ), and Precision of the proposed method

Analytes BBP DBP

Retention time (min) 12 .33 14 .19Linear range (μg L

_1) 5-500 10-500R2 0 .9985 0 .9990LOD (μg L

_1) 0 . 25 0 .52LOQ (μg L

_1) 0 . 83 1 .75RSD (%)Intra-day, %, n=7

10 μg L_1 4 .0 2 .9

100 μg L_1 3 . 8 3 .5

Inter-day, %, n =7 10 μg L

_1 3 . 1 3 .2100 μg L

_1 3 . 1 2 .9Recovery (%)Sample 1 . canned chicken in pepper oil

Spiking level of 2 μg kg_1 (RSD, %) 92 (4 .4) 87 (7 .5)

Spiking level of 10 μg kg_1 (RSD, %) 96 (5 .4) 90 (6 .6)

Samples 2 . plastic packaged chicken in sauceSpiking level of 2 μg kg

_1 (RSD, %) 86 (10 .1) 93 (7 .9)Spiking level of 10 μg kg

_1 (RSD, %) 88 (9 .1) 98 (5 .9)

Table 4 . Comparison of the proposed method with other methods

Method LOD of DBP and BBP (μg kg_1) Reference

GC-MS 0 .18‒2 .70 Cariou et al . (2016)SPE-GC 1 .21‒2 .51 Cinelli et al . (2014)SPE-HPLC-MS 1 .6–9 .8 Hayasaka (2014)UA-IL-DLLME-HPLC 0 .52 and 0 .25 This work

Note: GC -MS-gas chromatography mass spectrometrySPE-GC-solid phase extraction gas chromatographySPE-HPLC-MS-solid phase extraction liquid chromatography mass spectrometry

E.-Q. Xia et al.238

canned duck meat and plastic packaged duck meat. The analytical results are listed in Table 5.

From the real samples determination, the level of BBP and DBP was found less than 4.18 ± 0.31 μg kg

_1 and 2.12 ± 0.13 μg kg

_1, respect ively . The presence of analytes and their concentrations in various samples were significant distinction. The same results was also reported, and it indicated that different kind of PAEs were added in different packages, or other sources contributed analytes to food (Feng et al., 2016). The results showed similar level to other observations reported by different analytical methods (Sakhi et al., 2014; Holderbeke et al., 2014). The results obtained may not show any significant risk for the human health due to all concentration of analytes below relative national limited level. In addition, no significant increase level of the analytes released for the same samples during 9 months after production dates. Therefore, it is difficult to give enough information to track the migration of each PAE from packaging into food container attached. Maybe, a large number of the samples should be involved for the objective.

ConclusionsThe study developed a method based on ultrasound-assisted

QuEChERS combining with IL-DLLME coupled to HPLC for determining BBP and DBP in canned and plastic packaging solid food samples. It was verified efficient method for the target analytes with satisfied characteristics, such as precision less than RSD 4.0%, detection limits low to level obtained by expensive MS detector (between 0.25 and 0.52 μg L

_1) and good recovery above 86% with RSD less than 10.1%. Compared with other extraction technologies published, there have been the following advantages of quick, easy operation, less consumption of toxic solvents and time-saving for the present method. Furthermore, aided by QuEChERS and RSM, the efficiency of IL-DLLME was notably

improved in treatment of the complex solid food sample. The analytical results of real samples were below the national limited levels. In addition, only a slight increase of target compounds was found for the same products during 9 months after production date. A large number of samples must be involved in tracing the evidence of migration event in the further study.

Acknowledgements This work was supported by grants from the Doctoral Scientific Research Foundation of Guangdong Medical University (XB13010), and grant from Dongguan Key Laboratory of Environmental Medicine.

Conflict of interestThe authors declare that they have no conflict of interest.

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