Microemulsion electrokinetic chromatography for analysis of phthalates in soft drinks

Food Chemistry 141 (2013) 3486–3491

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Microemulsion electrokinetic chromatography for analysis of phthalates in soft drinks Sung-Yu Hsieh, Chun-Chi Wang, Shou-Mei Wu ⇑ School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan

a r t i c l e

i n f o

Article history: Received 24 November 2012 Received in revised form 5 April 2013 Accepted 6 June 2013 Available online 15 June 2013 Keywords: Phthalate Microemulsion electrokinetic chromatography PluronicÒ F-127

a b s t r a c t Microemulsion electrokinetic chromatography (MEEKC) is proposed for analysis of di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) in soft drinks. However, the instability of microemulsion is a critical issue. In this research, a novel material, PluronicÒ F-127, which has the properties of polymer and surfactant, was added for stabilizing the microemulsion in the MEEKC system. Our data demonstrate that the presence of PluronicÒ F-127 (0.05–0.30%) also helps enhance resolution of highly hydrophobic compounds, DBP and DEHP. The electrokinetic injection of sodium dodecyl sulphate (SDS) including sample ( 10 kV, 20 s) was introduced in this MEEKC system and this yielded about 25-fold sensitivity enhancement compared with hydrodynamic injection (1 psi, 10 s). During method validation, calibration curves were linear (r P 0.99), within a range of 75–500 ng/mL for DBP and 150–1000 ng/mL for DEHP. As the precision and accuracy assays, absolute values of relative standard deviation (RSD) and relative error (RE) in intraday (n = 3) and interday (n = 5) observations were less than 4.93%. This method was further applied for analyzing six commercial soft drinks and one was found containing 453.67 ng/mL of DEHP. This method is considered feasible for serving as a tool for analysis of highly hydrophobic molecules. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Phthalates are mainly used as plasticizers to increase flexibility, transparency, durability and longevity of plastics. Phthalates are easily released into the environment because there is no covalent bond between the phthalates and plastics in which they are mixed. People are commonly exposed to phthalates due to common use of plastics. It has been well documented that endocrine disruptors such as phthalates can be additive, so even very small amounts can interact with other chemicals to have cumulative, adverse ‘‘cocktail effects’’ (Waring & Harris, 2011). Capillary electrophoresis (CE) has developed very fast over the past two decades as a powerful analytical technique because of its high separation efficiency, high resolution, short analysis time and the small quantities of samples and reagents it requires. Micellar electrokinetic chromatography (MEKC) is one of the most useful CE modes and is often applied for analyzing neutral compounds by adding surfactants to form the micelles (Terabe, 2008, 2009). According to the ability of partition within the micelles, neutral analytes can be resolved. However, it is difficult to separate analytes possessing very high hydrophobicity by MEKC because all analytes are included by micelles. Therefore, microemulsion elec⇑ Corresponding author. Address: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: +886 73121101x2164; fax: +886 73159597. E-mail address: [email protected] (S.-M. Wu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.06.023

trokinetic chromatography (MEEKC) was developed for analyzing the highly hydrophobic compounds (Hefnawy et al., 2010; Huang, Lin, & Hsieh, 2009; Ryan, McEvoy, Donegan, Power, & Altria, 2011). Stable microemulsions have been used for delivery of food or drugs (Gharibzahedi, Razavi, & Mousavi, 2013; Rodea-González et al., 2012). Nevertheless, in MEEKC system, it is used to include the hydrophobic compound and to separate the analytes. MEEKC has been extensively used in many fields, such as food, pharmaceutical, clinical, biological and environmental analysis (Chen, Lin, Zhang, Cai, & Zhang, 2012; Miola, Snowden, & Altria, 1998; Piepel et al., 2012). It has been considered as an extension of MEKC (Huang, Lien, & Chiu, 2005). The mechanism of separation by MEEKC is similar to MEKC but the pseudostationary phases are in two different modes. In MEKC, micelle is formed by adding enough amount of surfactant above the critical micelle concentration (CMC). In MEEKC, the microemulsion consists of surfactants, oil and cosurfactants, where the cosurfactant stabilizes the nanometer-sized oil droplets by reducing the interfacial tension between the oil droplet and the buffer solution (Cao & Sheng, 2010; Ryan, Donegan, Power, & Altria, 2010). Therefore, cosurfactants play an important role in the formation of microemulsion. Several materials have been tried as cosurfactants, such as 1-butaol, 1-hexanol or 2-propnaol (Ryan et al., 2011). PluronicÒ F-127 (average MW 12,600), a three-block copolymer, consists of two hydrophilic ethylene oxide (EO) blocks and a central hydrophobic propylene oxide (PO) block (structure is

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EO106–PO70–EO106). The PluronicÒ F-127 possesses the properties of thermoresponsive polymers and polyoxyethylated nonionic surfactant. The self-aggregation in aqueous solutions to form micelles has resulted in its applications in several fields. Its CMC is 2.8  10 6 M and HLB is 22. Until now, it has been widely used in pharmaceutical technology, or has served as the matrix for analysis of DNA, proteins and peptides due to its properties of PluronicÒ F-127 micelles. (Ganguly, Kuperkar, Parekh, Aswal, & Bahadur, 2012; Krízek, Coufal, Tesarová, Sobotníková, & Bosáková, 2010; Liu et al., 2007; Pepic´, Hafner, Lovric´, Pirkic´, & Filipovic´-Grcic´, 2010). This material, when used as a cosurfactant, was expected to increase the efficiency of separation in MEEKC system. To the best of our knowledge, employment of copolymer PluronicÒ F-127 as cosurfactant for stabilizing MEEKC systems has not been reported previously. Therefore, we hope addition of the novel material PluronicÒ F-127 can effectively increase the resolution of the MEEKC system. Additionally, in order to improve the disadvantage of low sensitivity in CE analysis, many on-line stacking strategies have been developed, such as field-amplified sample injection (FASI) (Claude, Nehmé, & Morin, 2011; Zhang & Thormann, 1998), large volume sample stacking (LVSS) (Chien & Burgi, 1992; Mikkers, Everaerts, & Verheggen, 1979) or sweeping (Quirino & Terabe, 1998a; Wang, Han, Wang, Zang, & Wu, 2006). The sweeping method employs micelles or microemulsions to include the dispersive samples in capillary and can effectively improve CE sensitivity (Quirino & Terabe, 1998b). In this research, a MEEKC system combined with the sweeping method is developed for analysis of two highly hydrophobic compounds, di-n-butyl phthalate (DBP) and the longbranched di-(2-ethylhexyl) phthalate (DEHP), which are commonly found to be present in the environment, food products and plastics (Feng, Zhua, & Sensenstein, 2005; Frederiksen, Skakkebaek, & Andersson, 2007; Su et al., 2010). Suitability of the method as a feasible tool for detection of such highly hydrophobic analytes in commercial products, such as drinks, foods and cosmetics was examined.

2. Experimental 2.1. Materials Dibutyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP) were purchased from Chem Service (West Chester, USA). Sodium dihydrogen phosphate (NaH2PO4), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium dodecyl sulphate (SDS), ultra-pure water (grade of chromatography), n-octane and 1-butanol were purchased from Merck (Darmstadt, Germany). PluronicÒ F-127 and progesterone (as internal standard, IS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol was purchased from J. T. Baker (Philipsburg, NJ, USA). All chemical reagents were analytical grade.

2.2. CE system This experiment was performed on Beckman P/ACE MDQ instrument equipped with a UV detector (Beckman Instruments, Fullerton, CA, USA) and wavelength was set at 214 nm. The capillary (total length 40 cm; 30 cm to the detector) was uncoated fused-silica of 50 lm i.d. (Polymicro Technologies, Phoenix, AZ, USA). The temperature of the capillary was set at 25 °C. The new capillary was preconditioned by rinsing in methanol, 1 M HCl, 0.1 M NaOH and water. Between runs, the capillary was conditioned with MeOH at 40 psi for 5 min, water at 40 psi for 5 min and microemulsion buffer at 35 psi for 10 min.

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2.3. Preparation of microemulsion buffer and determination of particle size Microemulsion buffer for MEEKC was prepared by mixing 3.46% (w/w) SDS, 0.5% (w/w) n-octane, 7% (w/w) 1-butanol, 0.25% (w/w) PluronicÒ F-127 and 78.69% (w/w) of 30 mM NaH2PO4 (pH 2.5), sonicated by using BRANSON 5510R-DTH sonicator (BRANSON ULTRASONICS Co., Danbury, USA) for 30 min, and then 10% (v/v) MeOH was added into the solution with stirring for 5 min to obtain a stable and optically transparent microemulsion solution. After complete preparation, the microemulsion buffer was filtered through a 0.45-lm filter to remove particulate matter. The particle size distribution of microemulsions containing PluronicÒ F-127 was determined at room temperature by using Zetasizer 3000 HSA within 12 h. 3 mL of microemulsion solution was loaded in cuvette. The experiment was performed three times for each sample. 2.4. MEEKC sweeping system The standard stock solutions of DBP and DEHP were prepared in methanol with concentration of 0.5 mg/mL and were stored at 4 °C. Identical concentration of the mixture containing 5.0 lg/mL of IS was obtained by properly diluting the stock samples in 10 mM SDS solution, electrokinetically injected ( 10 kV for 20 s) into the capillary which had already been filled with microemulsion buffer. Finally, separation was accomplished at reverse polarity of 20 kV (anode at outlet end). Commercial sports drinks samples bought from local market were analyzed by this method. After 10-fold dilution in SDS solution, the drink samples were directly injected into the capillary without purification. All drink samples ready for analysis also contained 5.0 lg/mL of IS. 2.5. Method validation Progesterone (5 lg/mL) was used as an internal standard (IS) for quantitative analysis of DBP and DEHP in the MEEKC sweeping system. The range of calibration curve of DBP was 75–500 ng/mL, and it was 150–1000 ng/mL for DEHP. Precision and accuracy assays were conducted at concentrations of 150, 300 and 400 ng/mL for DBP, and 250, 400 and 800 ng/mL for DEHP. Validations were conducted by way of intraday (n = 3) and interday (n = 5) assays. Limits of detection were determined according to the concentration with signal to noise ratio of 3 (S/N = 3). 2.6. LC–MS system The LC–MS was performed on the Waters 2695 Separations Module–Waters micromass ZQ (Waters corporation, USA). The elution column was ZORBAX Eclipse Plus C18 Narrow Bore RR (2.1  100 mm, 3.5 lm). The mobile phase comprised 0.1% formic acid in H2O and 0.1% formic acid in methanol in equal proportions and the flow rate was 0.2 mL/min. This analysis was performed in the positive electrospray mode. The conditions of mass spectrometer were: voltage of ESI was 3 kV in capillary, 15 V in the cone, 3 V in the extractor and 0.1 V in the RF lens; temperatures of source and desolvation were 98 °C and 248 °C, respectively; and the gas flow rate was 600 L/h in desolvation and 100 L/h in the cone. 3. Results and discussion 3.1. Effect of PluronicÒ F-127 in MEEKC system and its particle size PluronicÒ F-127 was originally applied to capillary gel electrophoretical separation of oligonucleotides. Nevertheless, it also

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possesses the properties of surfactant and has been widely used as a cosurfactant for separating hydrophobic compounds. In this research, PluronicÒ F-127 was added to improve the resolution of the microemulsion in MEEKC. About 0.0–0.3% of PluronicÒ F-127 was added to the microemulsion system to evaluate the resolution of DBP and DEHP. The data are as shown in Fig. 1. When the microemulsion buffer had no PluronicÒ F-127, peaks of DBP and DEHP were coupled (Fig. 1A). Importantly, existence of PluronicÒ F-127 improved the resolution of the two hydrophobic compounds (Fig. 1B–E). We suggest that three blocks of PluronicÒ F-127 in the microemulsion can improve the hydrophobic selectivity with the analytes resulting in separation of DBP and DEHP. The selectivity indicates the ability of the microemulsion with or without addition of PluronicÒ F-127 to resolve the hydrophobic compounds of DBP and DEHP in CE. When the PluronicÒ F-127 was not added in the preparation of microemulsion, the DBP and DEHP could not be separated in this MEEKC system. Nevertheless, they could be differentiated by the existence of PluronicÒ F-127. The finding demonstrated the existence of PluronicÒ F-127 indeed increases the selective ability of the microemulsion. Because the log P of DEHP is larger than DBP (DBP: 4.72; DEHP: 7.45), DEHP highly interacts with the microemulsion of PluronicÒ F-127 and moves faster. Finally, 0.25% (w/w) PluronicÒ F-127 added in MEEKC system was selected as the optimal amount. The size of the droplet of the microemulsion containing 0.25% PluronicÒ F-127 ranged from 4.7 ± 0.4 nm to 5.7 ± 1.5 nm within

IS

12 h. (Table 1). No significant difference in the droplet size of the microemulsion was observed. The data of the droplet size supported the notion that the microemulsion was stable for 12 h. which was long enough for detection of samples. 3.2. Effect of organic modifiers in preparation of microemulsion Organic modifiers can effectively improve solubility of oil in microemulsion system. Therefore, in this research, two organic solvents of MeOH and 1-butanol were investigated. First, different amounts (3–11% w/w) of 1-butanol, which is also used as a cosurfactant in the preparation of the microemulsion, were used to investigate the separation efficiency (Fig. 2A). The peaks of DBP and DEHP could not be resolved by adding 3% 1-butanol. When the concentration of 1-butanol was above 7%, all analytes were separated. However, separation time increased at higher concentration and, therefore, 7% 1-butanol was treated as the optimal concentration. The organic modifier of MeOH is often used to improve the resolution in capillary electrophoresis. Here, different concentrations (0–15% v/v) of MeOH were added to the microemulsion system (Fig. 2B). DBP and DEHP were well separated at concentration of above 10% MeOH. When 15% MeOH was used, the interfered peak was coupled with the peak of DEHP. Finally, the analysis was performed by adding 10% MeOH. However, larger amounts of MeOH reduced the separation time. It is expected that the addition of MeOH effectively improves the retention factor and thus the separation time of highly hydrophobic compounds in MEEKC is shortened (Alexandridis, Holzwarthf, & Hatton, 1994). 3.3. Electrokinetic injection of SDS including samples in MEEKC

DBP DEHP

(E) 0.30%

(D) 0.25%

(C) 0.15%

(B) 0.05% 0.002 AU

In order to obtain large sensitivity enhancement, the highly hydrophobic analytes, DBP, DEHP and IS included in SDS micelle solution were electrokinetically injected. Because the critical micelle concentration of SDS is 8.1 mM, concentrations of the SDS solution from 8.0 to 25.0 mM were examined. When concentrations of SDS higher than 10 mM were used, significantly shorter peaks appeared (data not shown) because of the competitive injection of the extra SDS in the procedure of electrokinetic sample introduction. Finally, 10 mM SDS was chosen as the matrix for sample dilution. The injection time was also investigated ( 10 kV for 10–50 s). Longer injection time was followed by broad peaks (data not shown) resulting from the dispersion of SDS (high electrophoretic mobility), which included the samples. At last, the injection was accomplished at 10 kV for 20 s. Comparison between electrokinetic injection ( 10 kV, 20 s) and hydrodynamic injection (1 psi, 10 s) showed that sensitivity enhancement was 13.04-fold in DBP and 25.32-fold in DEHP. The electrokinetic injection of the highly hydrophobic compounds included in SDS micelles, equipped with MEEKC sweeping analysis, efficiently increased the detection sensitivity. Electropherograms of the two different injection modes are as shown in Fig. 3.

(A) 0.00%

0

5

10

15

20

Time (min) Fig. 1. Effect of Pluronic F-127 cosurfactant ratios (w/w) from 0.0% to 0.30% on separation of the two hydrophobic phthalate esters. Other experimental conditions: microemulsion system, 3.46% (w/w) SDS, 7% (w/w) 1-butanol, 0.5% (w/w) n-octane, 10% (w/v) MeOH and 78.69% (w/w) 30 mM NaH2PO4 buffer (pH 2.5); separation voltage, 20 kV; detection at 214 nm; electrokinetic injection, 10 kV for 20 s; concentrations of DBP and DEHP were 0.5 and 1 lg/mL, respectively. The IS was progesterone (5 lg/mL).

Table 1 The droplet size of microemulsion by adding PluronicÒ F-127 within 12 h. Time (h)

Droplet size (nm)

Polydispersity index

0 3 6 9 12

5.4 ± 1.3 4.7 ± 0.8 4.7 ± 0.4 5.7 ± 1.5 5.5 ± 0.4

0.15 ± 0.05 0.12 ± 0.06 0.13 ± 0.07 0.12 ± 0.07 0.19 ± 0.06

The determination of droplet size was performed in replicates of three times for each sample (n = 3).

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(A) Butanol

(B) MeOH

IS

DEHP

DBP

11%

IS DBP DEHP 15%

9%

10%

7%

5%

5% 0.002 AU

0.002 AU

0%

3% 0

5

10

15

20

25

0

5

10

15

20

Time (min)

Time (min)

Fig. 2. Effects of (A) 1-butanol ratios (w/w) from 3% to 11% and (B) MeOH ratios (w/w) from 0% to 12% in preparation of microemulsion for separation of DBP and DEHP. Other experimental conditions were as shown in Fig. 1.

Table 3 Precision and accuracy assays of DBP and DEHP in intraday and interday analysis.

DBP DEHP 0.001 AU MEEKC, inject -10 kV, 20s

DBP DEHP

10

12

MEEKC, inject, 1 psi, 10s

14

16

18

20

Time (min) Fig. 3. Comparison of MEEKC with pressure (1 psi, 10 s) and electrokinetic injection ( 10 kV, 20 s). Concentrations of DBP and DEHP were 0.5 and 1 lg/mL, respectively. Other experimental conditions were as shown in Fig. 1.

Intraday analysis (n = 3) DBP

DEHP

Interday analysis (n = 5) DBP

DEHP

Concentration known (ng/mL)

Concentration found (ng/mL)

RSD (%)

RE (%)

150.00 300.00 400.00 250.00 400.00 800.00

153.20 ± 6.09 300.11 ± 12.55 385.89 ± 6.28 249.58 ± 3.60 419.72 ± 13.75 777.21 ± 16.75

3.97 4.18 1.63 1.44 3.28 2.16

2.13 0.04 3.53 0.17 4.93 2.85

150.00 300.00 400.00 250.00 400.00 800.00

149.95 ± 3.16 295.09 ± 13.86 401.84 ± 15.43 258.07 ± 11.87 390.66 ± 12.33 804.52 ± 17.75

2.10 4.70 3.84 4.60 3.16 2.21

0.03 1.64 0.46 3.23 2.34 0.56

3.4. Method validation of MEEKC In this MEEKC sweeping system, two hydrophobic analytes, DBP and DEHP, were quantitatively determined by using progesterone (5 lg/mL) as the internal standard (IS). The calibration curves were linear over the range of 75–500 ng/mL for DBP, and 150–1000 ng/

mL for DEHP. Results of the linear regression analysis in intraday (n = 3) and interday (n = 5) assays were as shown in Table 2. Values of all coefficients of correlation were above 0.99 indicating good linearity of DBP and DEHP in this MEEKC sweeping system. Concentrations of DBP for precision and accuracy assays were

Table 2 Regression analysis of DBP and DEHP in intraday and interday assays. Concentration range (ng/mL)

Regression equation

Coefficient of correlation (r)

Intraday analysis (n = 3) DBP (75.0–500.0) DEHP (150–1000)

Y = (0.0007 ± 0.0000)X + (0.0102 ± 0.0058) Y = (0.0003 ± 0.0000)X + (0.0102 ± 0.0255)

0.99 0.99

Interday analysis (n = 5) DBP (75.0–500.0) DEHP (150–1000)

Y = (0.0068 ± 0.0004)X + (0.0139 ± 0.0034) Y = (0.0003 ± 0.0000)X + (0.0139 ± 0.0078)

0.99 0.99

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IS

(A) 0.008 DBP 0.007 DEHP 0.006

Spiked with DBP

0.005

Spiked with DEHP

0.004 0.003

Drink sample

0.002 10

12

14

16

18

Time (min)

(B) 20120522_Wu_Phthalate_New_6

2012/5/22

08:46:09

RT: 0.00 - 60.00 NL: 8.15E7 m/z= 391.28391.29 MS 20120522_ Wu_Phthalat e_New_6

21.36

100

18.93

90

Relative Abundance

80 70 60 21.52

50

22.12

40 30 20

22.29

10

2.63 4.43 6.79

9.35

0 0

5

10

12.68

23.41 25.02 28.39

13.19 15

20

25

31.79

35.08

30

35

38.35

42.05 43.22 40

55.55 55.86 56.65

48.72

45

50

55

Time (min) 20120522_Wu_Phthalate_New_6 #2090 RT: 19.25 AV: 1 NL: 5.12E7 T: FTMS + p NSI Full ms [100.00-1000.00] 391.28424 100 90

Relative Abundance

80 70 60 50 40 798.58826

30 20 10 0 100

354.33667 282.27899 261.14841 310.31036

149.02316 200

300

408.31036 400

672.55609

508.51981 563.55048 500

600

700

832.24091 776.60388 800

873.75439 920.48486 900

1000

m/z

Fig. 4. (A) Electropherogram of a real sports drink sample analyzed by this MEEKC sweeping system and (B) LC/MS for confirmation of DEHP in the drink sample. Concentration of DEHP in this sports drink was 453.67 ng/mL.

150, 300 and 400 ng/mL, and DEHP were 250, 400 and 800 ng/mL. All absolute values of relative standard deviation (RSD) and relative errors (RE) in intraday (n = 3) or interday (n = 5) assays were below 4.93% (Table 3). The data showed good precision and accuracy of

the MEEKC system. The limits of detection (LOD, S/N = 3) of DBP and DEHP were 25.0 and 50.0 ng/mL, respectively. In this MEEKC system, the microemulsion solution was freshly prepared before analysis, and the migration times of DEHP, DBP

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and IS were 12.56 ± 0.29, 12.94 ± 0.29 and 13.56 ± 0.33 min (all of the RSD < 2.4%), in the successive 10 runs, respectively. The data demonstrated that the microemulsion system provided stable conditions for analysis of DBP and DEHP within the effective time. 3.5. Applications This MEEKC sweeping method was applied for quantitative analysis of six commercial sports drinks. The real samples were directly electrokinetically injected into the capillary without pretreatment, after 10-fold dilution of the SDS micelle solution. In 5 sports drinks samples, neither DBP nor DEHP were detected. However, the existence of DEHP was detected in one sample. Electropherograms and the assay data are as shown in Fig. 4A. Concentration of DEHP in this drink sample was 453.67 ng/mL. The finding was confirmed by LC–MS (Fig. 4B) which demonstrates the drink sample indeed contained DEHP. The LC–MS was used only to confirm the existence of DEHP or DBP; it did not serve as a tool for quantification. This sample was also analyzed by a HPLC–UV method, and only DEHP was detected. However, concentration of DEHP could not be quantified (
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