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Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9
G Model
CCLET-2889; No. of Pages 5 Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9 Xiao-Lan Li a, Dong-Ling Meng a, Jiao Zhao b, Ya-Ling Yang a,* a b
Technology Centre of China Tobacco Guangxi Industrial Co., Ltd., Nanning 530001, China College of Life Science and Technology, Kunming University of Science and Technology, Kunming 650105, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2013 Received in revised form 12 January 2014 Accepted 26 February 2014 Available online xxx
This study aimed to establish a rapid analytical method to determine antioxidants in essence. A simple, efficient and practical, vortex-assisted, cloud-point extraction (VACPE) procedure is proposed for extracting and pre-concentrating four different of synthetic phenolic antioxidants (SPAs), (propyl gallate (PG), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)) in essence prior to high performance liquid chromatography (HPLC) analysis. The non-ionic surfactant, fatty alcohol polyoxyethylene ether-9 (AEO-9), was used as extractant and vortex-mixing was utilized to reduce extraction time and improve extraction efficiency. The effective parameters of the extraction process, such as volume of extraction solvent, pH, vortex-mixing time, equilibration temperature and time, were optimized. Under the optimum conditions, the linear range of PG, TBHQ, BHA and BHT was 8.0–800 ng/mL. All correlation coefficients of the calibration curves were higher than 0.996 and relative standard deviations (RSD, n = 5) were 2.36%–5.46%. The proposed method was successfully applied to the extraction and determination of antioxidants in essence samples with satisfactory relative recoveries of 89.4%–103.5%. The results confirmed the SPAs of essence could be effectively monitored by this method and also established good reference criteria for essence. ß 2014 Ya-Ling Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Vortex-assisted cloud-point extraction AEO-9 Antioxidant High performance liquid chromatography Essence
1. Introduction In compounding the complicated chemical elements of essence perfume, moderate amounts of solvents and other ingredients are utilized. In the process of production of essence and flavor, some synthetic phenolic antioxidants are added to prevent, or delay, the oxidation of the elements of essence, to improve stability and prolong the storage life [1]. A variety of detrimental factors can produce different degrees of oxidation of essence during the process of production and storage which seriously affect the quality of the essence and flavoring. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) tertbutyl hydroquinone (TBHQ) and propyl gallate (PG), etc. are most commonly used as synthetic phenolic antioxidants (SPAs). A large number of longterm experimental studies showed that consuming excessive quantities of synthetic antioxidants, to some extent, can cause damage to health. Normally, synthetic antioxidants are
* Corresponding author. E-mail address: [email protected] (Y.-L. Yang).
polyphenolic compounds that are more, or less, liposoluble, and suspected of being responsible for liver damage and carcinogenesis [2], usually at concentrations up to 100–200 mg/g in foods, either singly or in combination [3]. In the past, synthetic phenolic antioxidant testing mainly concentrated in the oil and grease of foods, or plastic product samples [4]. The analytical methods such as electrochemical analysis [5], high performance liquid chromatography [6,7], gas chromatography (GC) [8] and GC–MS [9,10] analysis, micellar electrokinetic capillary chromatography [11] and spectrophotometry [12] have been typically reported. Compared with other methods, high performance liquid chromatography has the advantages of more rapid analysis and high sensitivity. Sample preparation is usually necessary with liquid chromatography detection methods, to reduce, or eliminate, the complex, interfering matrices and concentration of the analyte is enriched. Therefore, an appropriate pretreatment method prior to chromatographic analysis has become more and more important. Liquid–liquid extraction (LLE) is the most common extraction method for the analysis of SPAs [13]. Other methods, such as solid phase extraction (SPE) [14], solid-phase microextraction (SPME)
http://dx.doi.org/10.1016/j.cclet.2014.03.005 1001-8417/ß 2014 Ya-Ling Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
G Model
CCLET-2889; No. of Pages 5 X.-L. Li et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
2
[15] and stir bar sorptive extraction (SBSE) [16] can be quite expensive. In this work, vortex-assisted, cloud-point extraction (VACPE) was used to reduce interference and enrich trace SPAs in essence when AEO-9 was used as the extraction solvent. AEO-9 does not exhibit background absorption in the ultraviolet region and fluorescence signals interfere with the determination of target analytes. The cloud-point temperature of AEO-9 is 30 8C similar to the Tergitol TMN series. Therefore, AEO-9 will have many advantages in cloud point extraction. Several factors affecting extraction were tested and to the best knowledge of the authors, the method is the first to be applied for determining SPAs in essence samples.
3. Results and discussion 3.1. Optimization of the CPE procedure In order to obtain the best analytical performance, the influence of different experimental parameters including the concentration of AEO-9, the equilibration temperature and time, the addition of (NH4)2SO4, sample pH, and vortex-mixing time on the performance of the CPE procedure were optimized. The recovery was used to evaluate the extraction efficiency under different experimental conditions. In this experiment, 5.0 mL of prepared flavor sample spiked with 500 ng/mL of each antioxidant was used for the study. All the experiments were performed five times and the averages of the results were used for optimization.
2. Experimental 3.2. Effect of sample pH 2.1. Chemical reagents All reagents were equal to, or above, analytical grade unless otherwise noted. LC-grade acetonitrile was purchased from Panreac (Barcelona, Spain) and fatty alcohol polyoxyethylene ether-9 (AEO-9) was purchased from Aladdin Chemical Co. (Shanghai, China). Ultra pure water was collected from a Milli-Q water purification system (Millipore, Madrid, Spain). Standards of TBHQ (99.0%), BHA (98.5%), PG (99.0%) and BHT (98%) were purchased from Aladdin Chemical Co. (Shanghai, China). Standard stock solutions of SPAs (TBHQ, BHA, PG and BHT) were prepared by dissolving in methanol at a concentration of 1000 mg/mL and stored in a freezer (4 8C) for 1 week. Working solutions were prepared by dilution of the stock solution with methanol. 2.2. Instrumentation and chromatographic conditions Sample analyses were performed on Agilent 1200 HPLC systems equipped with a vacuum degasser, an auto sampler, a quaternary pump, and a diode-array detector (DAD) (Agilent, Japan). Vortex agitator (Jiangsu, China) and water bath with temperature control (Shanghai, China) were used to implement extraction procedure. A centrifuge (Shanghai, China) was used for the phase separation process. Quantification was done by the evaluation of peak areas. Acetonitrile (A) and distilled-deionized water contained 0.1% acetic acid was used as mobile phase with the gradient program as follows: 0–4.5 min, 50%–85% A; 4.5–6.5 min, 85%–90%A; 6.5– 9.0 min, 90%–50% A. The flow rate was maintained at 1 mL/min. Column temperature was maintained at 25 8C and DAD detector was set at 280 nm during the entire process, each injection volume was 20 mL.
Because all the SPAs are weakly acidic compounds, in order to increase the extraction efficiencies of the SPAs in aqueous solution, the solution should be acidified to prevent them from dissociating during extraction. The effect of pH on the extraction efficiency of four antioxidants was studied over the pH range 3–11. As can be seen in Fig. 1, the highest signal intensity of antioxidants was obtained at pH 5.0. At a pH higher than 5, the extraction efficiencies decreased with pH, hence, pH 5 was selected for further experiments. 3.3. Effect of the equilibration temperature and time The shortest equilibration time and the lowest possible equilibration temperature are very desirable as a compromise between completion of extraction and efficient separation of phases. The dependence of extraction efficiency upon equilibrium temperature and time was studied over ranges of 25–45 8C and 5–30 min, respectively. The results showed that an equilibrium temperature of 30 8C is appropriate for the four antioxidants. Then, the extraction efficiency was nearly constant with the increase of time, and therefore 20 min was selected as the optimal extraction time. 3.4. Effect of nonionic surfactant volume AEO-9 was chosen as extraction solvent in VACPE because of the commercial availability in a highly-purified, homogenous form
80
Recovery (%)
2.3. Sample collection and pre-treatment The food flavors, obtained from spice enterprise companies in Naning and Hainan, were used for analysis and quantification of antioxidants. The flavor samples were stored in brown bottles and at 4 8C until analysis. 1 mL food flavor containing TBHQ, BHA, PG and BHT was placed in a screw-cap glass test tube with conical bottom and diluted to 5 mL with distilled-deionized water. After 0.1 mL AEO-9 (90%) and 0.2 g (NH4)2SO4 was added to the sample solution, the mixture was vortexed 30 s and followed by placing in a thermostat bath at 35 8C for 10 min, a cloudy solution was formed. After centrifugation at 4000 rpm for 10 min, the organic phase was diluted with acetonitrile to 0.2 mL, and filtered through 0.45 mm nylon syringe filter (Troody, China) in order to remove particles. An injection volume of 20.0 mL was injected into the HPLC instrument. A blank sample without any antioxidants was conducted as a reference.
BHT BHA TBHQ PG
100
60
40
20
0 2
4
6
8
10
12
pH Fig. 1. Effect of sample pH (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; AEO-9 volume 100 mL; n-octyl alcohol concentration,1.2%; equilibration temperature, 30 8C; equilibration time, 20 min; vortex-mixing time 30 s.).
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
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CCLET-2889; No. of Pages 5 X.-L. Li et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
TBHQ BHT BHA PG
100
TBHQ BHT BHA PG
100
80
Recovery (%)
90
Recovery (%)
3
80
70
60
60
40
20
50 80
90
100
110
120
0 0.8
1.2
1.6
2.0
2.4
2.8
n-octyl alcohol (%)
AEO-9 (uL) Fig. 2. Effect of the AEO-9 volume (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; sample pH, 5; n-octyl alcohol concentration, 1.2%; equilibration time, 20 min; equilibration temperature, 30 8C vortex-mixing time 30 s.).
Fig. 3. Effect of n-octyl alcohol concentration (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; sample pH 5; AEO-9 volume 100 mL; equilibration time, 20 min; equilibration temperature, 30 8C; vortex-mixing time 30 s.).
Table 1 Analytical parameters of the proposed method. Analyte
Regression equation
Linear range (ng/mL)
RSD (%, n = 5)
r2
Limit of detection (ng/mL)
PG TBHQ BHA BHT
y = 22.813x 0.259 y = 10.66x + 0.683 y = 11.96x + 0.6738 y = 16.66x + 0.6836
8–800 8–800 8–800 8–800
2.20 3.67 3.47 5.88
0.996 0.996 0.998 0.997
5.6 3.2 3.5 9.8
and low toxicological properties and its effective extraction of volatile substances. Furthermore, AEO-9 has no ultraviolet absorption in the UV area making it more suitable for use as an extracting agent for HPLC analysis. The pre-concentration efficiency was evaluated using AEO-9 concentrations ranging from 80 to 120 mL. The results are found in Fig. 2. A surfactant of volume 100 mL for AEO-9 was selected for all further work. 3.5. Effect of n-octyl alcohol concentration The cloud point of AEO-9 is 75 8C. In the presence of n-octyl alcohol, when very small quantities (less than 1.5%) are added to solutions of non-ionic surfactant, the cloud point decreases. However, with higher concentrations (more than 2.5%) of n-octyl alcohol, the solution phase splitting effect is poor. Organic alcohol has been noted to lower the cloud point due to organic alcohol molecules solubilized in the micelles of the barrier layer and leads to micelle inflation which reduces the cloud point of AEO-9.This phenomenon was noticed, in particular, by other authors [18]. Fig. 3 shows also that the addition of 1.2% n-octyl alcohol could lower cloud point and maximize the resulting recovery. It may be possible to have, for example, a separation of phases at room temperature. This can be of good utility in the extraction of products which are sensitive to heat, and may contribute to energy cost cutting for a process at an industrial scale.
30 s was selected for further experiment to emulsify the mixture completely. 3.7. Analytical characteristics Table 1 summarizes the analytical characteristics of the optimized method, such as regression equation, linear range, and limits of detection, reproducibility and enrichment factors. The linearity of the four antioxidants compounds was in the range 8.0– 800 ng/mL. The limits of detection (LODs) based on a signal-tonoise ratio (S/N) of 3 for PG, TBHQ, BHA, BHT were 5.6, 3.2, 3.5, 9.8 ng/mL. The relative standard deviation (RSD) of 500 ng/mL for PG, TBHQ, BHA, BHT was less than 5.88%. In addition, the experimental data indicated that the method has good ruggedness and could bear small variances. These parameters indicated that the present approach with high sensitivity and reliability could be used to detect the concentration of SPAs in essence. The typical HPLC chromatograms are shown in Fig. 4.
3.6. Effect of vortex-mixing time Vortex-mixing can accelerate the interactive rate between surfactant and aqueous phase so that the target analytes can be extracted at higher levels into surfactant-rich phase. Thus, different vortex time (15, 30, 60, 90, 120 s) on the extraction efficiency were evaluated. No significant effect was discovered when the vortex time ranged from 30 s to 120 s, which indicated that the mass transfer might be achieved in only 30 s. Therefore,
Fig. 4. Typical chromatograms of analysis of PG, TBHQ, BHA and BHT in essence samples by HPLC (B) and VACPE-HPLC (A) using AEO-9 as the extraction solvent.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
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Table 2 Accuracy of the method (relative recoveries with their RSDs) for sample solutions spiked at different concentrations. Analyte
Original (ng/mL)
Spiked (ng/mL)
Found (ng/mL)
Relative recovery (%)
RSD (n = 5, %)
PG
2.3
100 200 300
91.2 188.9 278.9
88.9 93.3 92.2
2.32 3.82 3.15
TBHQ
5.5
100 200 300
88.9 181.7 268.5
100.5 94.0 101.3
2.84 5.35 4.95
BHA
4.2
100 200 300
91.2 183.3 271.8
92.0 93.0 89.4
5.46 2.26 4.12
BHT
3.2
100 200 300
98.9 196.2 298.0
95.7 96.5 98.3
4.23 2.85 3.9
Table 3 Characteristic performance data obtained from CPE by using different surfactants for determination of SPAs. Surfactant
Extraction time
Equilibration temperature (8C)
Recovery (%)
LODa (ng/mL)
Ref.
Tergitol TMN-6 AES Triton X-114 Genapol-X080 AEO-9
40 min 15 min 30 min 40 min 30 min
45 90 37 55 30
90–98 – 98.9–101 89.5 89.4–103.5
1.6–9.0 – 3.6–6.3 – 3.2–9.8
[4] [17] [18] [19] This work
–: not mentioned.
3.8. Application In order to validate the viability of the proposed method in quantification of SPAs, the VACPE method was applied to the analysis of four SPAs in authentic essence samples, and at three different spiked levels (100.0, 200.0 and 300.0 ng/mL) of SPAs. Quantitative analyses of SPAs were performed using their peak areas and these areas were compared with the calibration graphs. The results are presented in Table 2. The recoveries determined on the addition of different concentrations of SPAs to samples are in the range of 89.4%–103.5%, with RSD (n = 5) of 2.26–5.35. The results show that the proposed method is effective for the determination of trace amounts of SPAs in the actual essence samples. 3.9. Comparison VACPE with other CPE methods The VACPE technique had been compared with other CPE methods using different surfactants, including Tergitol TMN-6 [4], AES [17], TritonX-114 [18] and Genapol-X080 [19]. The data are shown in Table 3. VACPE had a shorter extracting time and a lower equilibration time than other CPE methods. Recovery of AEO-9 was at least comparable to Tergitol TMN-6 and TritonX-114, and better than Genapol-X080. Equilibration temperature of AEO-9 was lower than other surfactants. Overall, AEO-9 can be developed as a new type of surfactant for cloud point extraction.
4. Conclusion In this report, vortex-assisted cloud-point extraction (VACPE) has been successfully used to quantify trace amounts of synthetic phenolic antioxidants, PG, TBHQ, BHA, BHT in essence samples using the HPLC technique coupled with DAD detector. AEO-9 is one of the most common, widespread surfactants and industrially available, non-ionic surfactants in a highly purified, homogenous form. Low toxicological properties are attributed to the absence of an aromatic ring in the molecular structure. The application of AEO-9 has the advantages of low temperature treatment, no
oxidizing deterioration to analytes, harmless to the health, stability and no ultraviolet absorption in the UV area. Based on these virtues, AEO-9 can be used as a new type of surfactant for cloud point extraction. To our knowledge this is the first report using AEO-9 as the extraction solvent in VACPE for the simultaneous determination of four different antioxidants in marketed essence samples. Therefore, VACPE is validated as a sensitive and simple microextraction method which has been successfully applied for the determination PG, TBHQ, BHA, BHT in essence samples. Acknowledgment This work was supported by College of Life Science and Technology, Kunming University of Science and Technology. References [1] J.X. Gong, L.J. Weng, F. Wang, et al., Synthesis and antioxidant properties of novel Silybin analogues, Chin. Chem. Lett. 17 (2006) 465–468. [2] H.C. Grice, Safety evaluation of butylated hydroxyanisole from the perspective of effects on forestomach and oesophageal squamous epithelium, Food Chem. Toxicol. 26 (1988) 717–723. [3] L. Chang, P.G. Bi, Y.N. Liu, et al., Simultaneous analysis of trace polymer additives in plastic beverage packaging by solvent sublation followed by high-performance liquid chromatography, J. Agric. Food Chem. 61 (2013) 7165–7171. [4] M. Chen, X.J. Hu, Z.G. Tai, et al., Determination of four synthetic phenolic antioxidants in edible oils by high-performance liquid chromatography with cloud point extraction using tergitol tmn-6, Food. Anal. Methods 6 (2013) 28–35. [5] Y.Q. Guan, Q.C. Chu, L. Fu, T. Wu, J.N. Ye, Determination of phenolic antioxidants by micellar electrokinetic capillary chromatography with electrochemical detection, Food Chem. 94 (2006) 157–162. [6] R.A. Medeiros, B.C. Lourenc¸a˜o, R.C. Rocha-Filho, O. Fatibello-Filho, Simple flow injection analysis system for simultaneous determination of phenolic antioxidants with multiple pulse amperometric detection at a boron-doped diamond electrode, Anal. Chem. 82 (2010) 8658–8663. [7] J. Karovicova, P. Simko, Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography, J. Chromatogr. A 882 (2000) 271– 281. [8] R. Rodil, J.B. Quintana, G. Basaglia, M.C. Pietrogrande, R. Cela, Determination of synthetic phenolic antioxidants and their metabolites in water samples by downscaled solid-phase extraction, silylation and gas chromatography-mass spectrometry, J. Chromatogr. A 1217 (2010) 6428–6435. [9] A. Zafra-Go´mez, B. Luzo´n-Toro, I. Jime´nez-Diaz, O. Ballesteros, A. Navalo´n, Quantification of phenolic antioxidants in rat cerebrospinal fluid by GC–MS after oral administration of compounds, J. Pharm. Biomed. Anal. 53 (2010) 103–108.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
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CCLET-2889; No. of Pages 5 X.-L. Li et al. / Chinese Chemical Letters xxx (2014) xxx–xxx [10] L. Guo, M.Y. Xie, A.P. Yan, Y.Q. Wan, Y.M. Wu, Simultaneous determination of five synthetic antioxidants in edible vegetable oil by GC–MS, Anal. Bioanal. Chem. 386 (2006) 1881–1887. [11] D. Djozan, Y. Assadi, Modified pencil lead as a new fiber for solid-phase microextraction, Chromatographia 60 (2004) 313–317. [12] X.L. Cao, A review recent development on analytical methods for determination of bisphenol a in food and biological samples, J. Liquid Chromatogr. Related Technol. 35 (2012) 2795–2829. [13] T.J. Klen, B.M. Vodopivec, Optimisation of olive oil phenol extraction conditions using a high-power probe ultrasonication, Food Chem. 134 (2012) 2481–2488. [14] A. Pukalskas, T.A. van Beek, P. de Waard, Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts, J. Chromatogr. A 1074 (2005) 81–88.
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[15] M. Mousavi, E. Noroozian, M. Jalali-Heravi, A. Mollahosseini, Optimization of solid-phase microextraction of volatile phenols in water by a polyaniline-coated Pt-fiber using experimental design, Anal. Chim. Acta 581 (2007) 71–77. [16] L. Montero, S. Conradi, S.H. Weiss, P. Popp, Determination of phenols in lake and ground water samples by stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry, J. Chromatogr. A 1071 (2005) 163–169. [17] M. Herrero, P.J. Martı´n-A´lvarez, F.J. Sen˜ora´ns, A. Cifuentes, E. Iba´n˜ez, Optimization of accelerated solvent extraction of antioxidants from Spirulina platensis microalga, Food Chem. 93 (2005) 417–423. [18] M. Chen, Q.H. Xia, M.S. Liu, Y.L. Yang, Cloud-point extraction and reversed-phase high-performance liquid chromatography for the determination of synthetic phenolic antioxidants in edible oils, J. Food Sci. 76 (2011) C98–C103. [19] O. Gortzi, S. Lalas, A. Chatzilazarou, et al., Recovery of natural antioxidants from olive mill wastewater using genapol-x080, J. Am. Oil Chem. Soc. 85 (2008) 133–140.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
CCLET-2889; No. of Pages 5 Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9 Xiao-Lan Li a, Dong-Ling Meng a, Jiao Zhao b, Ya-Ling Yang a,* a b
Technology Centre of China Tobacco Guangxi Industrial Co., Ltd., Nanning 530001, China College of Life Science and Technology, Kunming University of Science and Technology, Kunming 650105, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 November 2013 Received in revised form 12 January 2014 Accepted 26 February 2014 Available online xxx
This study aimed to establish a rapid analytical method to determine antioxidants in essence. A simple, efficient and practical, vortex-assisted, cloud-point extraction (VACPE) procedure is proposed for extracting and pre-concentrating four different of synthetic phenolic antioxidants (SPAs), (propyl gallate (PG), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)) in essence prior to high performance liquid chromatography (HPLC) analysis. The non-ionic surfactant, fatty alcohol polyoxyethylene ether-9 (AEO-9), was used as extractant and vortex-mixing was utilized to reduce extraction time and improve extraction efficiency. The effective parameters of the extraction process, such as volume of extraction solvent, pH, vortex-mixing time, equilibration temperature and time, were optimized. Under the optimum conditions, the linear range of PG, TBHQ, BHA and BHT was 8.0–800 ng/mL. All correlation coefficients of the calibration curves were higher than 0.996 and relative standard deviations (RSD, n = 5) were 2.36%–5.46%. The proposed method was successfully applied to the extraction and determination of antioxidants in essence samples with satisfactory relative recoveries of 89.4%–103.5%. The results confirmed the SPAs of essence could be effectively monitored by this method and also established good reference criteria for essence. ß 2014 Ya-Ling Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Vortex-assisted cloud-point extraction AEO-9 Antioxidant High performance liquid chromatography Essence
1. Introduction In compounding the complicated chemical elements of essence perfume, moderate amounts of solvents and other ingredients are utilized. In the process of production of essence and flavor, some synthetic phenolic antioxidants are added to prevent, or delay, the oxidation of the elements of essence, to improve stability and prolong the storage life [1]. A variety of detrimental factors can produce different degrees of oxidation of essence during the process of production and storage which seriously affect the quality of the essence and flavoring. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) tertbutyl hydroquinone (TBHQ) and propyl gallate (PG), etc. are most commonly used as synthetic phenolic antioxidants (SPAs). A large number of longterm experimental studies showed that consuming excessive quantities of synthetic antioxidants, to some extent, can cause damage to health. Normally, synthetic antioxidants are
* Corresponding author. E-mail address: [email protected] (Y.-L. Yang).
polyphenolic compounds that are more, or less, liposoluble, and suspected of being responsible for liver damage and carcinogenesis [2], usually at concentrations up to 100–200 mg/g in foods, either singly or in combination [3]. In the past, synthetic phenolic antioxidant testing mainly concentrated in the oil and grease of foods, or plastic product samples [4]. The analytical methods such as electrochemical analysis [5], high performance liquid chromatography [6,7], gas chromatography (GC) [8] and GC–MS [9,10] analysis, micellar electrokinetic capillary chromatography [11] and spectrophotometry [12] have been typically reported. Compared with other methods, high performance liquid chromatography has the advantages of more rapid analysis and high sensitivity. Sample preparation is usually necessary with liquid chromatography detection methods, to reduce, or eliminate, the complex, interfering matrices and concentration of the analyte is enriched. Therefore, an appropriate pretreatment method prior to chromatographic analysis has become more and more important. Liquid–liquid extraction (LLE) is the most common extraction method for the analysis of SPAs [13]. Other methods, such as solid phase extraction (SPE) [14], solid-phase microextraction (SPME)
http://dx.doi.org/10.1016/j.cclet.2014.03.005 1001-8417/ß 2014 Ya-Ling Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
G Model
CCLET-2889; No. of Pages 5 X.-L. Li et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
2
[15] and stir bar sorptive extraction (SBSE) [16] can be quite expensive. In this work, vortex-assisted, cloud-point extraction (VACPE) was used to reduce interference and enrich trace SPAs in essence when AEO-9 was used as the extraction solvent. AEO-9 does not exhibit background absorption in the ultraviolet region and fluorescence signals interfere with the determination of target analytes. The cloud-point temperature of AEO-9 is 30 8C similar to the Tergitol TMN series. Therefore, AEO-9 will have many advantages in cloud point extraction. Several factors affecting extraction were tested and to the best knowledge of the authors, the method is the first to be applied for determining SPAs in essence samples.
3. Results and discussion 3.1. Optimization of the CPE procedure In order to obtain the best analytical performance, the influence of different experimental parameters including the concentration of AEO-9, the equilibration temperature and time, the addition of (NH4)2SO4, sample pH, and vortex-mixing time on the performance of the CPE procedure were optimized. The recovery was used to evaluate the extraction efficiency under different experimental conditions. In this experiment, 5.0 mL of prepared flavor sample spiked with 500 ng/mL of each antioxidant was used for the study. All the experiments were performed five times and the averages of the results were used for optimization.
2. Experimental 3.2. Effect of sample pH 2.1. Chemical reagents All reagents were equal to, or above, analytical grade unless otherwise noted. LC-grade acetonitrile was purchased from Panreac (Barcelona, Spain) and fatty alcohol polyoxyethylene ether-9 (AEO-9) was purchased from Aladdin Chemical Co. (Shanghai, China). Ultra pure water was collected from a Milli-Q water purification system (Millipore, Madrid, Spain). Standards of TBHQ (99.0%), BHA (98.5%), PG (99.0%) and BHT (98%) were purchased from Aladdin Chemical Co. (Shanghai, China). Standard stock solutions of SPAs (TBHQ, BHA, PG and BHT) were prepared by dissolving in methanol at a concentration of 1000 mg/mL and stored in a freezer (4 8C) for 1 week. Working solutions were prepared by dilution of the stock solution with methanol. 2.2. Instrumentation and chromatographic conditions Sample analyses were performed on Agilent 1200 HPLC systems equipped with a vacuum degasser, an auto sampler, a quaternary pump, and a diode-array detector (DAD) (Agilent, Japan). Vortex agitator (Jiangsu, China) and water bath with temperature control (Shanghai, China) were used to implement extraction procedure. A centrifuge (Shanghai, China) was used for the phase separation process. Quantification was done by the evaluation of peak areas. Acetonitrile (A) and distilled-deionized water contained 0.1% acetic acid was used as mobile phase with the gradient program as follows: 0–4.5 min, 50%–85% A; 4.5–6.5 min, 85%–90%A; 6.5– 9.0 min, 90%–50% A. The flow rate was maintained at 1 mL/min. Column temperature was maintained at 25 8C and DAD detector was set at 280 nm during the entire process, each injection volume was 20 mL.
Because all the SPAs are weakly acidic compounds, in order to increase the extraction efficiencies of the SPAs in aqueous solution, the solution should be acidified to prevent them from dissociating during extraction. The effect of pH on the extraction efficiency of four antioxidants was studied over the pH range 3–11. As can be seen in Fig. 1, the highest signal intensity of antioxidants was obtained at pH 5.0. At a pH higher than 5, the extraction efficiencies decreased with pH, hence, pH 5 was selected for further experiments. 3.3. Effect of the equilibration temperature and time The shortest equilibration time and the lowest possible equilibration temperature are very desirable as a compromise between completion of extraction and efficient separation of phases. The dependence of extraction efficiency upon equilibrium temperature and time was studied over ranges of 25–45 8C and 5–30 min, respectively. The results showed that an equilibrium temperature of 30 8C is appropriate for the four antioxidants. Then, the extraction efficiency was nearly constant with the increase of time, and therefore 20 min was selected as the optimal extraction time. 3.4. Effect of nonionic surfactant volume AEO-9 was chosen as extraction solvent in VACPE because of the commercial availability in a highly-purified, homogenous form
80
Recovery (%)
2.3. Sample collection and pre-treatment The food flavors, obtained from spice enterprise companies in Naning and Hainan, were used for analysis and quantification of antioxidants. The flavor samples were stored in brown bottles and at 4 8C until analysis. 1 mL food flavor containing TBHQ, BHA, PG and BHT was placed in a screw-cap glass test tube with conical bottom and diluted to 5 mL with distilled-deionized water. After 0.1 mL AEO-9 (90%) and 0.2 g (NH4)2SO4 was added to the sample solution, the mixture was vortexed 30 s and followed by placing in a thermostat bath at 35 8C for 10 min, a cloudy solution was formed. After centrifugation at 4000 rpm for 10 min, the organic phase was diluted with acetonitrile to 0.2 mL, and filtered through 0.45 mm nylon syringe filter (Troody, China) in order to remove particles. An injection volume of 20.0 mL was injected into the HPLC instrument. A blank sample without any antioxidants was conducted as a reference.
BHT BHA TBHQ PG
100
60
40
20
0 2
4
6
8
10
12
pH Fig. 1. Effect of sample pH (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; AEO-9 volume 100 mL; n-octyl alcohol concentration,1.2%; equilibration temperature, 30 8C; equilibration time, 20 min; vortex-mixing time 30 s.).
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
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TBHQ BHT BHA PG
100
TBHQ BHT BHA PG
100
80
Recovery (%)
90
Recovery (%)
3
80
70
60
60
40
20
50 80
90
100
110
120
0 0.8
1.2
1.6
2.0
2.4
2.8
n-octyl alcohol (%)
AEO-9 (uL) Fig. 2. Effect of the AEO-9 volume (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; sample pH, 5; n-octyl alcohol concentration, 1.2%; equilibration time, 20 min; equilibration temperature, 30 8C vortex-mixing time 30 s.).
Fig. 3. Effect of n-octyl alcohol concentration (extraction conditions: sample volume, 5.0 mL spiked with 500 ng/mL of each antioxidant; sample pH 5; AEO-9 volume 100 mL; equilibration time, 20 min; equilibration temperature, 30 8C; vortex-mixing time 30 s.).
Table 1 Analytical parameters of the proposed method. Analyte
Regression equation
Linear range (ng/mL)
RSD (%, n = 5)
r2
Limit of detection (ng/mL)
PG TBHQ BHA BHT
y = 22.813x 0.259 y = 10.66x + 0.683 y = 11.96x + 0.6738 y = 16.66x + 0.6836
8–800 8–800 8–800 8–800
2.20 3.67 3.47 5.88
0.996 0.996 0.998 0.997
5.6 3.2 3.5 9.8
and low toxicological properties and its effective extraction of volatile substances. Furthermore, AEO-9 has no ultraviolet absorption in the UV area making it more suitable for use as an extracting agent for HPLC analysis. The pre-concentration efficiency was evaluated using AEO-9 concentrations ranging from 80 to 120 mL. The results are found in Fig. 2. A surfactant of volume 100 mL for AEO-9 was selected for all further work. 3.5. Effect of n-octyl alcohol concentration The cloud point of AEO-9 is 75 8C. In the presence of n-octyl alcohol, when very small quantities (less than 1.5%) are added to solutions of non-ionic surfactant, the cloud point decreases. However, with higher concentrations (more than 2.5%) of n-octyl alcohol, the solution phase splitting effect is poor. Organic alcohol has been noted to lower the cloud point due to organic alcohol molecules solubilized in the micelles of the barrier layer and leads to micelle inflation which reduces the cloud point of AEO-9.This phenomenon was noticed, in particular, by other authors [18]. Fig. 3 shows also that the addition of 1.2% n-octyl alcohol could lower cloud point and maximize the resulting recovery. It may be possible to have, for example, a separation of phases at room temperature. This can be of good utility in the extraction of products which are sensitive to heat, and may contribute to energy cost cutting for a process at an industrial scale.
30 s was selected for further experiment to emulsify the mixture completely. 3.7. Analytical characteristics Table 1 summarizes the analytical characteristics of the optimized method, such as regression equation, linear range, and limits of detection, reproducibility and enrichment factors. The linearity of the four antioxidants compounds was in the range 8.0– 800 ng/mL. The limits of detection (LODs) based on a signal-tonoise ratio (S/N) of 3 for PG, TBHQ, BHA, BHT were 5.6, 3.2, 3.5, 9.8 ng/mL. The relative standard deviation (RSD) of 500 ng/mL for PG, TBHQ, BHA, BHT was less than 5.88%. In addition, the experimental data indicated that the method has good ruggedness and could bear small variances. These parameters indicated that the present approach with high sensitivity and reliability could be used to detect the concentration of SPAs in essence. The typical HPLC chromatograms are shown in Fig. 4.
3.6. Effect of vortex-mixing time Vortex-mixing can accelerate the interactive rate between surfactant and aqueous phase so that the target analytes can be extracted at higher levels into surfactant-rich phase. Thus, different vortex time (15, 30, 60, 90, 120 s) on the extraction efficiency were evaluated. No significant effect was discovered when the vortex time ranged from 30 s to 120 s, which indicated that the mass transfer might be achieved in only 30 s. Therefore,
Fig. 4. Typical chromatograms of analysis of PG, TBHQ, BHA and BHT in essence samples by HPLC (B) and VACPE-HPLC (A) using AEO-9 as the extraction solvent.
Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005
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Table 2 Accuracy of the method (relative recoveries with their RSDs) for sample solutions spiked at different concentrations. Analyte
Original (ng/mL)
Spiked (ng/mL)
Found (ng/mL)
Relative recovery (%)
RSD (n = 5, %)
PG
2.3
100 200 300
91.2 188.9 278.9
88.9 93.3 92.2
2.32 3.82 3.15
TBHQ
5.5
100 200 300
88.9 181.7 268.5
100.5 94.0 101.3
2.84 5.35 4.95
BHA
4.2
100 200 300
91.2 183.3 271.8
92.0 93.0 89.4
5.46 2.26 4.12
BHT
3.2
100 200 300
98.9 196.2 298.0
95.7 96.5 98.3
4.23 2.85 3.9
Table 3 Characteristic performance data obtained from CPE by using different surfactants for determination of SPAs. Surfactant
Extraction time
Equilibration temperature (8C)
Recovery (%)
LODa (ng/mL)
Ref.
Tergitol TMN-6 AES Triton X-114 Genapol-X080 AEO-9
40 min 15 min 30 min 40 min 30 min
45 90 37 55 30
90–98 – 98.9–101 89.5 89.4–103.5
1.6–9.0 – 3.6–6.3 – 3.2–9.8
[4] [17] [18] [19] This work
–: not mentioned.
3.8. Application In order to validate the viability of the proposed method in quantification of SPAs, the VACPE method was applied to the analysis of four SPAs in authentic essence samples, and at three different spiked levels (100.0, 200.0 and 300.0 ng/mL) of SPAs. Quantitative analyses of SPAs were performed using their peak areas and these areas were compared with the calibration graphs. The results are presented in Table 2. The recoveries determined on the addition of different concentrations of SPAs to samples are in the range of 89.4%–103.5%, with RSD (n = 5) of 2.26–5.35. The results show that the proposed method is effective for the determination of trace amounts of SPAs in the actual essence samples. 3.9. Comparison VACPE with other CPE methods The VACPE technique had been compared with other CPE methods using different surfactants, including Tergitol TMN-6 [4], AES [17], TritonX-114 [18] and Genapol-X080 [19]. The data are shown in Table 3. VACPE had a shorter extracting time and a lower equilibration time than other CPE methods. Recovery of AEO-9 was at least comparable to Tergitol TMN-6 and TritonX-114, and better than Genapol-X080. Equilibration temperature of AEO-9 was lower than other surfactants. Overall, AEO-9 can be developed as a new type of surfactant for cloud point extraction.
4. Conclusion In this report, vortex-assisted cloud-point extraction (VACPE) has been successfully used to quantify trace amounts of synthetic phenolic antioxidants, PG, TBHQ, BHA, BHT in essence samples using the HPLC technique coupled with DAD detector. AEO-9 is one of the most common, widespread surfactants and industrially available, non-ionic surfactants in a highly purified, homogenous form. Low toxicological properties are attributed to the absence of an aromatic ring in the molecular structure. The application of AEO-9 has the advantages of low temperature treatment, no
oxidizing deterioration to analytes, harmless to the health, stability and no ultraviolet absorption in the UV area. Based on these virtues, AEO-9 can be used as a new type of surfactant for cloud point extraction. To our knowledge this is the first report using AEO-9 as the extraction solvent in VACPE for the simultaneous determination of four different antioxidants in marketed essence samples. Therefore, VACPE is validated as a sensitive and simple microextraction method which has been successfully applied for the determination PG, TBHQ, BHA, BHT in essence samples. Acknowledgment This work was supported by College of Life Science and Technology, Kunming University of Science and Technology. References [1] J.X. Gong, L.J. Weng, F. Wang, et al., Synthesis and antioxidant properties of novel Silybin analogues, Chin. Chem. Lett. 17 (2006) 465–468. [2] H.C. Grice, Safety evaluation of butylated hydroxyanisole from the perspective of effects on forestomach and oesophageal squamous epithelium, Food Chem. Toxicol. 26 (1988) 717–723. [3] L. Chang, P.G. Bi, Y.N. Liu, et al., Simultaneous analysis of trace polymer additives in plastic beverage packaging by solvent sublation followed by high-performance liquid chromatography, J. Agric. Food Chem. 61 (2013) 7165–7171. [4] M. Chen, X.J. Hu, Z.G. Tai, et al., Determination of four synthetic phenolic antioxidants in edible oils by high-performance liquid chromatography with cloud point extraction using tergitol tmn-6, Food. Anal. Methods 6 (2013) 28–35. [5] Y.Q. Guan, Q.C. Chu, L. Fu, T. Wu, J.N. Ye, Determination of phenolic antioxidants by micellar electrokinetic capillary chromatography with electrochemical detection, Food Chem. 94 (2006) 157–162. [6] R.A. Medeiros, B.C. Lourenc¸a˜o, R.C. Rocha-Filho, O. Fatibello-Filho, Simple flow injection analysis system for simultaneous determination of phenolic antioxidants with multiple pulse amperometric detection at a boron-doped diamond electrode, Anal. Chem. 82 (2010) 8658–8663. [7] J. Karovicova, P. Simko, Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography, J. Chromatogr. A 882 (2000) 271– 281. [8] R. Rodil, J.B. Quintana, G. Basaglia, M.C. Pietrogrande, R. Cela, Determination of synthetic phenolic antioxidants and their metabolites in water samples by downscaled solid-phase extraction, silylation and gas chromatography-mass spectrometry, J. Chromatogr. A 1217 (2010) 6428–6435. [9] A. Zafra-Go´mez, B. Luzo´n-Toro, I. Jime´nez-Diaz, O. Ballesteros, A. Navalo´n, Quantification of phenolic antioxidants in rat cerebrospinal fluid by GC–MS after oral administration of compounds, J. Pharm. Biomed. Anal. 53 (2010) 103–108.
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Please cite this article in press as: X.-L. Li, et al., Determination of synthetic phenolic antioxidants in essence perfume by high performance liquid chromatography with vortex-assisted, cloud-point extraction using AEO-9, Chin. Chem. Lett. (2014), http:// dx.doi.org/10.1016/j.cclet.2014.03.005