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Single-drop microextraction combined with gas chromatography-electron capture detection for the determination of acrylamide in food samples
Food Chemistry 274 (2019) 55–60
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Analytical Methods
Single-drop microextraction combined with gas chromatography-electron capture detection for the determination of acrylamide in food samples
T
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Mohammad Saraji , Salman Javadian Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Gas chromatography-electron capture detection Single-drop microextraction Derivatization Food sample Acrylamide
A single-drop microextraction method followed by gas chromatography-electron capture detection was developed to determine acrylamide in food samples. Acrylamide was extracted by water and derivatized by hydrobromic acid in the presence of ammonium peroxydisulfate. The derivatization was carried out at 45 °C in 15 min using 46 µL of hydrobromic acid and 98 mg of ammonium peroxydisulfate. A 3.0-mL volume of the derivatized analyte was extracted using a 1.0-µL n-octanol droplet hanging from the needle tip of a GC microsyringe. After extraction, the extract was injected into the gas chromatograph. The influence of experimental parameters effective on derivatization reaction yield and extraction performance was studied. The limit of detection and quantification, relative standard deviation and linearity of the method were 0.60 µg/L, 2.0 µg/L, < 6.0%, and 2.0–100.0 µg/L, respectively. The method was utilized to determine acrylamide in three food samples (i.e., bread, potato chips and cookie).
1. Introduction Acrylamide is a low molecular weight compound with high solubility in water. It is very reactive in air and is rapidly polymerized. Acrylamide is commercially produced by the reaction between sulfuric acid and acrylonitrile, or the hydration of acrylonitrile using a copper catalyst (Asano, Yasuda, Tani, & Yamada, 1982; Smith & Oehme, 1991). Formation of acrylamide in carbohydrate-rich foods (e.g., bread, biscuit, potato chips, rice, and corn) at elevated temperatures was discovered by a group of Swedish researchers in 2002 (Zhang, Zhang, & Zhang, 2005). The main mechanism of the procedure is the Maillard reaction, in which asparagine amino acid is converted to acrylamide in the presence of reducing sugars such as glucose (Zhang et al., 2005). Acrylamide is distributed throughout the human body after entering blood (Lopachin & Gavin, 2008), damages the neuron system (Matthäus, Haase, & Vosmann, 2004), and causes infertility, eye infection and weakness of irritability (Brathen & Knutsen, 2005; Lignert et al., 2002). It is also known as a carcinogenic compound (Besaratinia & Pfeifer, 2007). The tolerable daily intake for the carcinogenicity and neurotoxicity of acrylamide is 2.6 and 40 µg per day for each Kg of body weight, respectively (Tardiff, Gargas, Kirman, Leigh Carson, & Sweeney, 2010). Due to the daily consumption of carbohydrate-rich foods by human, determining the concentration level of acrylamide in these samples is
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very important. The analytical instruments used for determining acrylamide include gas chromatography-electron capture detection (GCECD) (Hashimoto, 1976; Zhang, Dong, Ren, & Zhang, 2006), GC-mass spectrometry (MS) (Weisshaar, 2004), liquid chromatography (LC) with ultraviolet (UV) (Geng, Jiang, & Chen, 2008; Shi, Zhang, & Zhao, 2009; Wang, Lee, Shuang, & Choi, 2008), or MS (Rufianhenares & Morales, 2006; Weisshaar, 2004) detection, and capillary electrophoresis (CE) with UV and MS (Bermudo, Nnuez, Puignou, & Galceran, 2006; Bermudo, Nnuez, Moyano, Puignou, & Galceran, 2007; Oracz, Nebesny, & Zyzelewicz, 2011). Because of the low sensitivity of LC-UV and CEUV for the determination of acrylamide, it should be derivatized before detection (Bermudo et al., 2006; Shi et al., 2009; Oracz et al., 2011). Among the different instruments, GC has been widely applied for acrylamide determination. Measuring acrylamide concentration in food samples, without derivatization by GC, has been reported in a few articles (Kaykhaii & Abdi, 2013; Weisshaar, 2004). However, to improve the extraction capability of the analyte and increase the sensitivity of analysis, a derivatization step is necessary before GC analysis. For derivatization, acrylamide is generally brominated to 2,3-dibromopropanamide (2,3-DBPA) (Areke, Ydberg, Arlsson, & Riksson, 2002; Hashimoto, 1976; Pittet, Perisset, & Oberson, 2004). In almost all studies, a very toxic reagent (Br2) has been used for brominating acrylamide, and the derivatization process was time-consuming. In the standard EPA method, the mixture of KBr, HBr and Br2 (Hashimoto,
Corresponding author. E-mail address: [email protected] (M. Saraji).
https://doi.org/10.1016/j.foodchem.2018.08.108 Received 17 January 2018; Received in revised form 14 July 2018; Accepted 24 August 2018 Available online 25 August 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
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head column pressure of 0.1 MPa was used as the carrier gas. A DB1701 GC column (30 m, 0.25 mm I.D. and 0.15 µm thicknesses) from Agilent Technologies (Palo Alto, CA, USA) was used for separation. Injection port and detector temperatures were set at 240 and 280 °C, respectively. All injections were performed in split mode at the split ratio of 1/18. Column temperature programming was as follow: 60 °C for 1.0 min, then increased by 12 °C/min until 150 °C (2 min hold), and finally, increased to 170 °C (2 min hold) at the rate of 30 °C/min.
1976; US Environmental Protection Agency, 1996) is used for derivatization. Due to the use of very toxic reagents in this procedure, a mixture of KBr and KBrO3 has also been employed for derivatizing acrylamide to avoid using bromine (Zhang et al., 2006; Zhang, Ren, Zhao, & Zhang, 2007). However, the reaction yield for producing 2,3DBPA was < 5%. Hence, another reaction product (i.e., 2-bromopropenamide) with the reaction yield > 95% was used for quantification. As a disadvantage, the amount of the derivatization reagents should be carefully adjusted because it has a great effect on the derivatization reaction yield (Zhang et al., 2006; Zhang et al., 2007). In addition, the extract should be cleaned before instrumental analysis. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) have mainly been applied for extracting derivatized acrylamide (Areke et al., 2002; Hashimoto, 1976; US Environmental Protection Agency 1996; Zhang et al., 2005; Zhang et al., 2006; Zhang et al., 2007). LLE and SPE are tedious procedures that use large volumes of expensive and noxious solvents. Today, solvent microextraction techniques as simple, inexpensive and environmentally friendly methods have attracted researchers’ attention for sample preparation. Only two studies have reported the analysis of acrylamide in food samples using microextraction techniques (Kaykhaii & Abdi, 2013; Qu, Liu, Luo, Qiu, & Chen, 2013). In the first report, the analyte in an aqueous solution was derivatized using a derivatization procedure by KBr/KBrO3. The derivatized analyte (mono bromide propanamide) was extracted by a solid-phase microextraction fiber (Qu et al., 2013). In the second study, acrylamide was extracted from potato chips by an organic solvent (200 mL ethyl hexanoate), followed by single-drop microextraction (SDME) (Kaykhaii & Abdi, 2013). The procedure is not environmentally friendly due to using a large volume of ethyl hexanoate. In the present study, SDME followed by GC-ECD detection was used for extracting and determining acrylamide in food samples. A novel and more environmentally friendly derivatization procedure was used to derivatize acrylamide to 2,3-DBPA. A mixture of HBr and (NH4)2S2O8 was used to perform the derivatization reaction at a relatively low temperature (45 °C). Extraction solvent and derivatization reagents were used at micro amounts. The parameters affecting derivatization yield and extraction reactions were studied and optimized. The analytical performance and feasibility of the method for analyzing food samples were also studied.
2.3. Derivatization procedure A 3.0-mL standard aqueous solution of acrylamide (100.0 µg/L), 98 mg of ammonium peroxydisulfate and 46 µL of HBr were added to an 8-mL glass vial. The glass vial was sealed with a polyethylene cap and heated (at 45 °C) in a water bath on a magnetic stirrer for 15 min. The solution was then neutralized by adding NaOH solution (1.5 mol/L). To ensure complete neutralization process, the pH of the solution was tested by a pH paper. The solution was diluted with water in a 5.0 mL volumetric flask and used for SDME. 2.4. SDME procedure A 10.0 µL GC syringe (Hamilton, Bonaduz, Switzerland) was employed to perform the SDME experiments. To perform SDME, 3.0 mL solution containing the derivatized analyte, 1.0 g NaCl, and a stir bar were added into a 5-mL extraction vial. The vial was placed on a magnetic stirrer (MR 3000D, Heidolph, Germany). A volume of 1.0 µL of n-octanol was withdrawn into the syringe and the syringe needle immersed into the solution. The plunger was depressed, and a 1.0-µL solvent drop was formed at the tip of the needle. The solution was stirred at the rate of 300 rpm for 5.0 min. After extraction, the organic solvent containing the extracted analyte was retracted into the syringe and analyzed by GC-ECD. 2.5. Real samples To investigate the applicability of the present method in complex matrices, different food samples including bread, potato chips and cookie were analyzed by the method. The samples were provided from a local supermarket. They were dried in a heating oven (50 °C) for one night. After that, the samples were grinded by a metal mortar. An amount between 2 and 3 g of the grinded sample was transferred into a beaker. In the case of cookie and potato chips samples, a 3-mL volume of n-hexane was added, and the solution agitated on a shaker (Aria Teb Co., Tehran, Iran) for 5 min. After that, the sample was centrifuged, and the n-hexane phase was discarded. The fat extraction with n-hexane was repeated one more time. The sample was dried at room temperature. A volume of 100 mL of water was added to the sample and the solution ultrasonicated for 20 min. The clear solution was then appropriately diluted (10 times for cookie and bread samples and 5 times for potato chips) with water, and 3 mL of the diluted sample was used for performing the derivatization process. For spiking the samples, a standard aqueous solution of 100 mg/L of acrylamide was used. The samples were spiked at two concentration levels. Bread sample (3.0 g) was spiked with 90 and 150 µL of the standard solution of acrylamide. For potato chips, 40 and 80 µL of the acrylamide solution was added to the samples. Cookie sample was spiked with 80 and 160 µL of the acrylamide solution. Each experiment was replicated three times to calculate the relative standard deviation (RSD) of data. To assess the accuracy of the method, real samples were spiked with the different amounts of acrylamide and spiking recovery was calculated. The spiking recovery was defined as:
2. Experimental 2.1. Chemicals and reagents Pure acrylamide and 2,3-DBPA were purchased from Merck (Darmstadt, Germany) and Alfa Aesar (Ward Hill, MA, USA), respectively. GC grade n-octanol was obtained from Fluka (Buchs, Switzerland). Methanol and HPLC grade hexane were purchased from Caledon Laboratories (Georgetown, Ont., Canada). Hydrobromic acid (48%) was obtained from Daejung Co. (Siheung, Korea). Ammonium peroxydisulfate, sodium chloride, and sodium hydroxide were also obtained from Merck. Pure water was prepared by a water purification system consisted of ion exchange and carbon cartridges (Overseas Equipment & Services, OK, USA). Stock standard solutions of acrylamide and 2,3-DBPA were prepared at the concentration of 1000 mg/L in water and methanol, respectively. A solution at the concentration of 10.0 mg/L was prepared by diluting the standard solutions with water. Diluted working solutions were prepared daily from the above standard solution. To protect the standard solutions from light, the containers were covered with aluminum foil and kept in a refrigerator. 2.2. Instrumentation
spiking recovery (%) = A gas chromatograph (BFRL, Beijing, China), model 3420 equipped with ECD was utilized. Nitrogen gas with the purity of 99.999% at a
Cfound−Creal Cadded
× 100
where Cfound, Creal, and Cadded are the concentration of analyte measured 56
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in the sample after the addition of a known amount of acrylamide, the concentration of analyte measured in the real sample before spiking, and the amount of acrylamide added to the sample, respectively. 3. Results and discussion 3.1. Derivatization of acrylamide Oxidative bromination with hydrogen peroxide has previously been applied for brominating carbonyl containing compounds such as ketones and diketones without the need for a catalyst (Podgorsek, Stavber, Zupan, & Iskra, 2007). Bromine is in-situ prepared through oxidation of HBr using H2O2 (Eq. (1)). A 1:1 to 2:1 mol ratio of H2O2:HBr gives the best conversion yield for the α-bromination of different carbonyl compounds (Podgorsek et al., 2007). In the present work, the same derivatization reaction was used to produce 2,3-DBPA by brominating acrylamide (Eq. (2)).
2HBr + H2 O2 → Br2 + 2H2 O
(1)
Br2 + H2 C= CH−CONH2 → H2 CBr−CHBr−CONH2
(2)
Fig. 2. Influence of the reaction time on the derivatization reaction yield. Concentration of acrylamide: 100.0 µg/L, sample volume: 3.0 mL, derivatization temperature: 45 °C.
the reaction yield did not change significantly (Fig. 2). According to the results, the optimum condition was derivatization of 3 mL sample solution at 45 °C for 15 min using 98 mg of (NH4)2S2O8 (as oxidant) and 46 µL of HBr. The reagents used in this method have less toxicity than bromine, which is directly used for the bromination of acrylamide, as reported in previous studies. In addition, the amount of the reagents consumed in this method was at the micro scale compared to the mL (or g) range commonly used in other techniques (Areke et al., 2002; Yamini, Ghambarian, & Esrafili, 2012; Zhang et al., 2006; Zhang et al., 2007). Moreover, the derivatization process presented in the method is relatively fast.
To find out the best derivatization reaction efficiency, different parameters affecting the conversion yield of acrylamide were investigated. A 3-mL aqueous standard solution of acrylamide at 100 µg/L was used. After derivatization, the amount of derivatized analyte was measured using SDME-GC-ECD (Section 2.4). The effect of different amounts of the reagents (i.e., oxidant and HBr) on derivatization efficiency was studied using two oxidants (H2O2 or (NH4)2S2O8). The mole ratio of oxidant:HBr was 1:1 in all experiments. Different mole ratios of reagents (oxidant:HBr) to the analyte from 103 to 105 were investigated. The results (data not shown) showed that the amount of the derivatized analyte was enhanced by increasing the amount of the reagents up to 105. At higher amounts of the reagents, the solvent drop was not stable due to significant changes in the chemical composition of the matrix. Thus, amounts higher than 105 were not evaluated. In addition, the analyte signal using (NH4)2S2O8 as the oxidant was almost 30% higher than that using H2O2. To find out the effect of reaction time and temperature, a standard aqueous solution of acrylamide (100 µg/L) was derivatized using (NH4)2S2O8:HBr at the mole ratio of 105 relative to the analyte. The sample was derivatized at different temperatures (4, 25, 35, 45, 54 and 74 °C) for 15 min. The results presented in Fig. 1 show that the maximum response was obtained at 45 °C, and at higher temperatures, the reaction yield remained constant. To investigate the effect of reaction time on the derivatization efficiency, the reaction times of 5.0, 10.0, 15.0, 30.0 and 60.0 min were tested at 45 °C. It was found that the highest derivatization efficiency was obtained in 15 min, and after that
3.2. SDME optimization To investigate the optimal conditions for the extraction process in SDME method, variables including the type of extraction solvent, extraction time, ionic strength of solution and solution temperature should be optimized. A 3.0-mL standard aqueous solution of 2,3-DBPA (100.0 µg/L) was used to study the above-mentioned extraction parameters on the SDME efficiency. All determinations were reported from the average of three replicate measurements. 3.2.1. Extraction solvent The type of organic solvent used for extracting analyte is an important factor in SDME method. The solvent should have a good affinity for the analyte, low solubility in water, be stable during the extraction process, and have good chromatographic behavior (Nagaraju & Huang, 2007). As indicated in Fig. 3, n-octanol was chosen as the best solvent for extracting analyte. It is to be noted that the 4:1 ratio (v/v) of hexane:ethylacetate, reported as the selected solvent in LLE of acrylamide, had about eight times lower efficiency than n-octanol (Areke et al., 2002; Fernandes & Soares, 2007; Pittet et al., 2004). 3.2.2. Salt addition In SDME method, the extraction of analyte can be increased or decreased by adding salt. The extraction yield can increase due to the salting out effect. However, in some cases, high salt concentrations limit the extraction since the diffusion rate of analyte into the organic phase decreases due to the changing physical properties of the solvent–water interface (Lopez-Blanco, Blanco-Cid, Cancho-Grande, & Simal-Gandara, 2003). To investigate the effect of salt addition on the extraction efficiency, different amounts of NaCl from 0 to 33% (w/v) were studied. According to the results (Fig. S1, Supplementary information), the saturated solution of NaCl (33%, w/v) yielded the best performance.
Fig. 1. Influence of the reaction temperature on the derivatization reaction yield. Concentration of acrylamide: 100.0 µg/L, sample volume: 3.0 mL, derivatization time: 15 min.
3.2.3. Extraction temperature At high temperatures, the mass transfer and diffusion of the analyte 57
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3.2.4. Extraction time Like other microextraction methods, SDME is an equilibrium–based process (Bahramifar, Yamini, Shariati-Feizabadi, & Shamsipur, 2002). To investigate the effect of time on the extraction of the target analyte, different times in the range of 0.5–50.0 min were examined. The results (Fig. S3, Supplementary information) showed that the maximum extraction was obtained in 5 min, and after that, the extraction efficiency remained constant. 3.3. Method validation To calculate the figures of merits of the method, a 3.0-mL standard solution of acrylamide was used. The optimized derivatization and extraction conditions (98 mg of (NH4)2S2O8, 46 L of HBr, derivatization time of 15.0 min at 45 °C, 1.0 µL of n-octanol, stirring rate of 300 rpm, 33% (w/v) NaCl and extraction time of 5.0 min at room temperature) were applied to determine acrylamide. The linearity of the method was in the range of 2.0–100.0 µg/L with the determination coefficient of 0.9984. The precision of the method obtained using standard solutions of the analyte at the concentration levels of 10.0, and 100.0 µg/L were 4.6 and 6.0%, respectively. The limit of detection and quantification calculated based on peak-to-peak noise (S/N = 3 and 10) were 0.60 and 2.0 µg/L, respectively. Some characteristics of the present method and other techniques used for the determination of acrylamide in food samples are listed in Table 2. The main feature of the present method, which is in the framework of green chemistry was very low consumption of extractant and derivatization reagents. Moreover, the method showed a low detection limit and short derivatization time
Fig. 3. Effect of different organic solvents on the extraction efficiency. Concentration of 2,3-dibromopropanamide: 100.0 µg/L, sample volume: 3.0 mL, solvent volume: 1.0 µL, solution temperature: 25 °C, sample pH: 7, extraction time: 10.0 min, NaCl concentration: 13.3%, w/v. Table 1 Analysis of real samples, recovery and accuracy data. Sample
Added (µg/g)
Founda (µg/g)
Recovery (%)
Potato chips
– 2.00 4.00
1.24 3.21 5.26
– 98 100
Bread
– 3.00 5.00
1.70 4.83 6.62
– 104 98
Cookie
– 4.00 8.00
1.41 5.36 9.42
– 97 99
a
3.4. Real sample analysis To evaluate the effect of matrix components on the quantification process and the method capability for analyzing real samples, the method was applied to determine acrylamide in bread, potato chips and cookie (with no creams and nuts). The standard addition method was used to quantify the analyte in the samples. The samples were spiked with the acrylamide standard solution at two concentration levels. The spiking levels were chosen based on the initial concentration of analyte in the real samples assessed by preliminary experiments. The low spiked concentration levels were 2.0, 3.0 and 4.0 µg/g for potato chips, bread, and cookie, respectively. For the high concentration level, potato chips, bread, and cookie were spiked at 4.0, 5.0 and 8.0 µg/g, respectively. There was a good linear relationship (r2 > 0.99) between the spiked amounts and the peak height of the analyte in the real samples (Fig. S4, Supplementary information). The amounts of acrylamide found in bread, potato chips and cookie were 1.7 ± 0.1, 1.24 ± 0.08
The relative standard deviations of data are between 1.5 and 9.5% (n = 3).
toward solvent drop is done faster. On the other hand, due to the increase in the solubility of organic solvent at higher temperatures and thus the decrease in the solvent volume, extraction efficiency diminishes. High temperatures can also make the drop unstable and deteriorate method repeatability (Jeannot & Cantwell, 1996). To study the effect of temperature on extraction yield, different temperatures from 15 to 50 °C were investigated. Considering the data (Fig. S2, Supplementary information), 25 °C had the best performance for analyte extraction, and consequently, the room temperature was chosen as the optimum extraction temperature.
Table 2 Analytical characteristics of different methods used for the determination of acrylamide in food samples. Method
Sample amount (g)
Derivatization reagent
Derivatization time (min)
Extraction solvent volume (mL)
LOD (µg/ L)
Reference
LLE-GC–MS
10.0
7.5 g KBr, 10.0 mL Br2, HBra
720
5
SPE-LC-MS SPE-LC-MS-MS
1.0 10.0
– –
– –
hexane:ethyl acetate (4:1, 40) H2O (4.0) + MeOH (1.0) ACN (1.0) + H2O (5.0)
23.2 10
SPE-LC-UV LLE-LC-UV LLE-GC-ECD
2.0 0.5 1.5
– 100 30
H2O (5.0) + MeOH (5.0) ethyl acetate (8.0) ethyl acetate (12.0)
30 15 0.1
SPME-GC-ECD
1.0
60
–
0.26
Yanqin Qu et al. (2013)
RP SDMEb-GC-FID SDME-GC-ECD
50.0 2.0–3.0
– 0.5 g 2-mercapto benzoic acid 1.5 g KBr, 1.0 mL KBrO3 (0.1 M) 1.0 g KBr, 1.0 mL KBrO3 (0.1 M) – 46 µL HBr, 98 mg (NH4)2S2O8
Rufianhenares and Morales (2006) Kaykhaii and Abdi (2013) Rufianhenares and Morales (2006) Oracz et al. (2011) Bermudo et al. (2006) Podgorsek et al. (2007)
– 15
H2O (3.5 × 10−3) n-octanol (1.0 × 10−3)
57 0.6
Kaykhaii and Abdi (2013) This work
a b
Acidified to pH 1–3. Reversed phase single-drop microextraction. 58
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Fig. 4. GC–ECD chromatograms obtained by derivatization with HBr and (NH4)2S2O8 followed by SDME of (a) unspiked potato chips and (b) potato chips spiked with 100.0 mg/L of acrylamide.
and 1.41 ± 0.11 µg/g, respectively. The spiked level and the measured amount of the analyte in the samples are listed in Table 1. As can be seen in Table 1, the spiking recovery was higher than 97%, demonstrating that the matrix components of the samples had no significant adverse effect on analyte quantification. The chromatograms of unspiked and spiked potato chips with the analyte at 4 µg/g are shown in Fig. 4.
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4. Conclusions The present SDME-GC-ECD method was successfully used to determine acrylamide in the foods. The method allowed detection of the analyte at sub-ppb concentration level. Acrylamide was simply extracted from the samples in 5.0 min, and a very small volume of the extractant (1.0 µL of n-octanol) was used. In comparison to the different derivatization methods used to determine acrylamide by GC, the toxic bromine reagent was not used in this method, and other reagents used for the derivatization of the analyte were consumed at micro amounts (46 µL of hydrobromic acid and 98 mg of ammonium peroxydisulfate). Moreover, the derivatization reaction time greatly decreased. Compared to the previously published methods for determining acrylamide in food samples, the present method as a green and environmentally friendly technique showed low detection limit, good precision, very short derivatization and extraction time, and low solvent, reagent and sample consumption. Acknowledgments The authors are grateful to the Research Council of Isfahan University of Technology (IUT, Iran) and Center of Excellence in Sensor and Green Chemistry (Iran) for the financial support of this project. Conflict of interest The authors declared no conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.foodchem.2018.08.108. References Areke, E. D. E. N. T., Ydberg, P. E. R. R., Arlsson, P. A. K., & Riksson, S. U. N. E. E. (2002). Analysis of acrylamide, a carcinogen formed in heated food stuffs. Journal of
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Rufianhenares, J., & Morales, F. (2006). Determination of acrylamide in potato chips by a reversed-phase LC–MS method based on a stable isotope dilution assay. Food Chemistry, 97, 555–562. Shi, Z., Zhang, H., & Zhao, X. (2009). Ultrasonic-assisted precolumn derivatization-HPLC determination of acrylamide formed in Radix Asparagi during heating process. Journal of Pharmaceutical and Biomedical Analysis, 49, 1045–1047. Smith, E. A., & Oehme, F. W. (1991). Acrylamide and polyacrylamide: A review of production, use, environmental fate and neurotoxicity. Reviews on Environmental Health, 9, 215–228. Tardiff, R. G., Gargas, M. L., Kirman, C. R., Leigh Carson, M., & Sweeney, L. M. (2010). Estimation of safe dietary intake levels of acrylamide for humans. Food and Chemical Toxicology, 48, 658–667. US Environmental Protection Agency (1996). Acrylamide by gas chromatography. Vol I. EPA 8032aWashington, DC: Office of Test Methods for Evaluating Solid Waste, Physical/Chemical Methods (SW-846). Wang, H., Lee, A. W. M., Shuang, S., & Choi, M. M. F. (2008). SPE/HPLC/UV studies on acrylamide in deep-fried flour-based indigenous Chinese foods. Microchemical
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Analytical Methods
Single-drop microextraction combined with gas chromatography-electron capture detection for the determination of acrylamide in food samples
T
⁎
Mohammad Saraji , Salman Javadian Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Gas chromatography-electron capture detection Single-drop microextraction Derivatization Food sample Acrylamide
A single-drop microextraction method followed by gas chromatography-electron capture detection was developed to determine acrylamide in food samples. Acrylamide was extracted by water and derivatized by hydrobromic acid in the presence of ammonium peroxydisulfate. The derivatization was carried out at 45 °C in 15 min using 46 µL of hydrobromic acid and 98 mg of ammonium peroxydisulfate. A 3.0-mL volume of the derivatized analyte was extracted using a 1.0-µL n-octanol droplet hanging from the needle tip of a GC microsyringe. After extraction, the extract was injected into the gas chromatograph. The influence of experimental parameters effective on derivatization reaction yield and extraction performance was studied. The limit of detection and quantification, relative standard deviation and linearity of the method were 0.60 µg/L, 2.0 µg/L, < 6.0%, and 2.0–100.0 µg/L, respectively. The method was utilized to determine acrylamide in three food samples (i.e., bread, potato chips and cookie).
1. Introduction Acrylamide is a low molecular weight compound with high solubility in water. It is very reactive in air and is rapidly polymerized. Acrylamide is commercially produced by the reaction between sulfuric acid and acrylonitrile, or the hydration of acrylonitrile using a copper catalyst (Asano, Yasuda, Tani, & Yamada, 1982; Smith & Oehme, 1991). Formation of acrylamide in carbohydrate-rich foods (e.g., bread, biscuit, potato chips, rice, and corn) at elevated temperatures was discovered by a group of Swedish researchers in 2002 (Zhang, Zhang, & Zhang, 2005). The main mechanism of the procedure is the Maillard reaction, in which asparagine amino acid is converted to acrylamide in the presence of reducing sugars such as glucose (Zhang et al., 2005). Acrylamide is distributed throughout the human body after entering blood (Lopachin & Gavin, 2008), damages the neuron system (Matthäus, Haase, & Vosmann, 2004), and causes infertility, eye infection and weakness of irritability (Brathen & Knutsen, 2005; Lignert et al., 2002). It is also known as a carcinogenic compound (Besaratinia & Pfeifer, 2007). The tolerable daily intake for the carcinogenicity and neurotoxicity of acrylamide is 2.6 and 40 µg per day for each Kg of body weight, respectively (Tardiff, Gargas, Kirman, Leigh Carson, & Sweeney, 2010). Due to the daily consumption of carbohydrate-rich foods by human, determining the concentration level of acrylamide in these samples is
⁎
very important. The analytical instruments used for determining acrylamide include gas chromatography-electron capture detection (GCECD) (Hashimoto, 1976; Zhang, Dong, Ren, & Zhang, 2006), GC-mass spectrometry (MS) (Weisshaar, 2004), liquid chromatography (LC) with ultraviolet (UV) (Geng, Jiang, & Chen, 2008; Shi, Zhang, & Zhao, 2009; Wang, Lee, Shuang, & Choi, 2008), or MS (Rufianhenares & Morales, 2006; Weisshaar, 2004) detection, and capillary electrophoresis (CE) with UV and MS (Bermudo, Nnuez, Puignou, & Galceran, 2006; Bermudo, Nnuez, Moyano, Puignou, & Galceran, 2007; Oracz, Nebesny, & Zyzelewicz, 2011). Because of the low sensitivity of LC-UV and CEUV for the determination of acrylamide, it should be derivatized before detection (Bermudo et al., 2006; Shi et al., 2009; Oracz et al., 2011). Among the different instruments, GC has been widely applied for acrylamide determination. Measuring acrylamide concentration in food samples, without derivatization by GC, has been reported in a few articles (Kaykhaii & Abdi, 2013; Weisshaar, 2004). However, to improve the extraction capability of the analyte and increase the sensitivity of analysis, a derivatization step is necessary before GC analysis. For derivatization, acrylamide is generally brominated to 2,3-dibromopropanamide (2,3-DBPA) (Areke, Ydberg, Arlsson, & Riksson, 2002; Hashimoto, 1976; Pittet, Perisset, & Oberson, 2004). In almost all studies, a very toxic reagent (Br2) has been used for brominating acrylamide, and the derivatization process was time-consuming. In the standard EPA method, the mixture of KBr, HBr and Br2 (Hashimoto,
Corresponding author. E-mail address: [email protected] (M. Saraji).
https://doi.org/10.1016/j.foodchem.2018.08.108 Received 17 January 2018; Received in revised form 14 July 2018; Accepted 24 August 2018 Available online 25 August 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
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head column pressure of 0.1 MPa was used as the carrier gas. A DB1701 GC column (30 m, 0.25 mm I.D. and 0.15 µm thicknesses) from Agilent Technologies (Palo Alto, CA, USA) was used for separation. Injection port and detector temperatures were set at 240 and 280 °C, respectively. All injections were performed in split mode at the split ratio of 1/18. Column temperature programming was as follow: 60 °C for 1.0 min, then increased by 12 °C/min until 150 °C (2 min hold), and finally, increased to 170 °C (2 min hold) at the rate of 30 °C/min.
1976; US Environmental Protection Agency, 1996) is used for derivatization. Due to the use of very toxic reagents in this procedure, a mixture of KBr and KBrO3 has also been employed for derivatizing acrylamide to avoid using bromine (Zhang et al., 2006; Zhang, Ren, Zhao, & Zhang, 2007). However, the reaction yield for producing 2,3DBPA was < 5%. Hence, another reaction product (i.e., 2-bromopropenamide) with the reaction yield > 95% was used for quantification. As a disadvantage, the amount of the derivatization reagents should be carefully adjusted because it has a great effect on the derivatization reaction yield (Zhang et al., 2006; Zhang et al., 2007). In addition, the extract should be cleaned before instrumental analysis. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) have mainly been applied for extracting derivatized acrylamide (Areke et al., 2002; Hashimoto, 1976; US Environmental Protection Agency 1996; Zhang et al., 2005; Zhang et al., 2006; Zhang et al., 2007). LLE and SPE are tedious procedures that use large volumes of expensive and noxious solvents. Today, solvent microextraction techniques as simple, inexpensive and environmentally friendly methods have attracted researchers’ attention for sample preparation. Only two studies have reported the analysis of acrylamide in food samples using microextraction techniques (Kaykhaii & Abdi, 2013; Qu, Liu, Luo, Qiu, & Chen, 2013). In the first report, the analyte in an aqueous solution was derivatized using a derivatization procedure by KBr/KBrO3. The derivatized analyte (mono bromide propanamide) was extracted by a solid-phase microextraction fiber (Qu et al., 2013). In the second study, acrylamide was extracted from potato chips by an organic solvent (200 mL ethyl hexanoate), followed by single-drop microextraction (SDME) (Kaykhaii & Abdi, 2013). The procedure is not environmentally friendly due to using a large volume of ethyl hexanoate. In the present study, SDME followed by GC-ECD detection was used for extracting and determining acrylamide in food samples. A novel and more environmentally friendly derivatization procedure was used to derivatize acrylamide to 2,3-DBPA. A mixture of HBr and (NH4)2S2O8 was used to perform the derivatization reaction at a relatively low temperature (45 °C). Extraction solvent and derivatization reagents were used at micro amounts. The parameters affecting derivatization yield and extraction reactions were studied and optimized. The analytical performance and feasibility of the method for analyzing food samples were also studied.
2.3. Derivatization procedure A 3.0-mL standard aqueous solution of acrylamide (100.0 µg/L), 98 mg of ammonium peroxydisulfate and 46 µL of HBr were added to an 8-mL glass vial. The glass vial was sealed with a polyethylene cap and heated (at 45 °C) in a water bath on a magnetic stirrer for 15 min. The solution was then neutralized by adding NaOH solution (1.5 mol/L). To ensure complete neutralization process, the pH of the solution was tested by a pH paper. The solution was diluted with water in a 5.0 mL volumetric flask and used for SDME. 2.4. SDME procedure A 10.0 µL GC syringe (Hamilton, Bonaduz, Switzerland) was employed to perform the SDME experiments. To perform SDME, 3.0 mL solution containing the derivatized analyte, 1.0 g NaCl, and a stir bar were added into a 5-mL extraction vial. The vial was placed on a magnetic stirrer (MR 3000D, Heidolph, Germany). A volume of 1.0 µL of n-octanol was withdrawn into the syringe and the syringe needle immersed into the solution. The plunger was depressed, and a 1.0-µL solvent drop was formed at the tip of the needle. The solution was stirred at the rate of 300 rpm for 5.0 min. After extraction, the organic solvent containing the extracted analyte was retracted into the syringe and analyzed by GC-ECD. 2.5. Real samples To investigate the applicability of the present method in complex matrices, different food samples including bread, potato chips and cookie were analyzed by the method. The samples were provided from a local supermarket. They were dried in a heating oven (50 °C) for one night. After that, the samples were grinded by a metal mortar. An amount between 2 and 3 g of the grinded sample was transferred into a beaker. In the case of cookie and potato chips samples, a 3-mL volume of n-hexane was added, and the solution agitated on a shaker (Aria Teb Co., Tehran, Iran) for 5 min. After that, the sample was centrifuged, and the n-hexane phase was discarded. The fat extraction with n-hexane was repeated one more time. The sample was dried at room temperature. A volume of 100 mL of water was added to the sample and the solution ultrasonicated for 20 min. The clear solution was then appropriately diluted (10 times for cookie and bread samples and 5 times for potato chips) with water, and 3 mL of the diluted sample was used for performing the derivatization process. For spiking the samples, a standard aqueous solution of 100 mg/L of acrylamide was used. The samples were spiked at two concentration levels. Bread sample (3.0 g) was spiked with 90 and 150 µL of the standard solution of acrylamide. For potato chips, 40 and 80 µL of the acrylamide solution was added to the samples. Cookie sample was spiked with 80 and 160 µL of the acrylamide solution. Each experiment was replicated three times to calculate the relative standard deviation (RSD) of data. To assess the accuracy of the method, real samples were spiked with the different amounts of acrylamide and spiking recovery was calculated. The spiking recovery was defined as:
2. Experimental 2.1. Chemicals and reagents Pure acrylamide and 2,3-DBPA were purchased from Merck (Darmstadt, Germany) and Alfa Aesar (Ward Hill, MA, USA), respectively. GC grade n-octanol was obtained from Fluka (Buchs, Switzerland). Methanol and HPLC grade hexane were purchased from Caledon Laboratories (Georgetown, Ont., Canada). Hydrobromic acid (48%) was obtained from Daejung Co. (Siheung, Korea). Ammonium peroxydisulfate, sodium chloride, and sodium hydroxide were also obtained from Merck. Pure water was prepared by a water purification system consisted of ion exchange and carbon cartridges (Overseas Equipment & Services, OK, USA). Stock standard solutions of acrylamide and 2,3-DBPA were prepared at the concentration of 1000 mg/L in water and methanol, respectively. A solution at the concentration of 10.0 mg/L was prepared by diluting the standard solutions with water. Diluted working solutions were prepared daily from the above standard solution. To protect the standard solutions from light, the containers were covered with aluminum foil and kept in a refrigerator. 2.2. Instrumentation
spiking recovery (%) = A gas chromatograph (BFRL, Beijing, China), model 3420 equipped with ECD was utilized. Nitrogen gas with the purity of 99.999% at a
Cfound−Creal Cadded
× 100
where Cfound, Creal, and Cadded are the concentration of analyte measured 56
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in the sample after the addition of a known amount of acrylamide, the concentration of analyte measured in the real sample before spiking, and the amount of acrylamide added to the sample, respectively. 3. Results and discussion 3.1. Derivatization of acrylamide Oxidative bromination with hydrogen peroxide has previously been applied for brominating carbonyl containing compounds such as ketones and diketones without the need for a catalyst (Podgorsek, Stavber, Zupan, & Iskra, 2007). Bromine is in-situ prepared through oxidation of HBr using H2O2 (Eq. (1)). A 1:1 to 2:1 mol ratio of H2O2:HBr gives the best conversion yield for the α-bromination of different carbonyl compounds (Podgorsek et al., 2007). In the present work, the same derivatization reaction was used to produce 2,3-DBPA by brominating acrylamide (Eq. (2)).
2HBr + H2 O2 → Br2 + 2H2 O
(1)
Br2 + H2 C= CH−CONH2 → H2 CBr−CHBr−CONH2
(2)
Fig. 2. Influence of the reaction time on the derivatization reaction yield. Concentration of acrylamide: 100.0 µg/L, sample volume: 3.0 mL, derivatization temperature: 45 °C.
the reaction yield did not change significantly (Fig. 2). According to the results, the optimum condition was derivatization of 3 mL sample solution at 45 °C for 15 min using 98 mg of (NH4)2S2O8 (as oxidant) and 46 µL of HBr. The reagents used in this method have less toxicity than bromine, which is directly used for the bromination of acrylamide, as reported in previous studies. In addition, the amount of the reagents consumed in this method was at the micro scale compared to the mL (or g) range commonly used in other techniques (Areke et al., 2002; Yamini, Ghambarian, & Esrafili, 2012; Zhang et al., 2006; Zhang et al., 2007). Moreover, the derivatization process presented in the method is relatively fast.
To find out the best derivatization reaction efficiency, different parameters affecting the conversion yield of acrylamide were investigated. A 3-mL aqueous standard solution of acrylamide at 100 µg/L was used. After derivatization, the amount of derivatized analyte was measured using SDME-GC-ECD (Section 2.4). The effect of different amounts of the reagents (i.e., oxidant and HBr) on derivatization efficiency was studied using two oxidants (H2O2 or (NH4)2S2O8). The mole ratio of oxidant:HBr was 1:1 in all experiments. Different mole ratios of reagents (oxidant:HBr) to the analyte from 103 to 105 were investigated. The results (data not shown) showed that the amount of the derivatized analyte was enhanced by increasing the amount of the reagents up to 105. At higher amounts of the reagents, the solvent drop was not stable due to significant changes in the chemical composition of the matrix. Thus, amounts higher than 105 were not evaluated. In addition, the analyte signal using (NH4)2S2O8 as the oxidant was almost 30% higher than that using H2O2. To find out the effect of reaction time and temperature, a standard aqueous solution of acrylamide (100 µg/L) was derivatized using (NH4)2S2O8:HBr at the mole ratio of 105 relative to the analyte. The sample was derivatized at different temperatures (4, 25, 35, 45, 54 and 74 °C) for 15 min. The results presented in Fig. 1 show that the maximum response was obtained at 45 °C, and at higher temperatures, the reaction yield remained constant. To investigate the effect of reaction time on the derivatization efficiency, the reaction times of 5.0, 10.0, 15.0, 30.0 and 60.0 min were tested at 45 °C. It was found that the highest derivatization efficiency was obtained in 15 min, and after that
3.2. SDME optimization To investigate the optimal conditions for the extraction process in SDME method, variables including the type of extraction solvent, extraction time, ionic strength of solution and solution temperature should be optimized. A 3.0-mL standard aqueous solution of 2,3-DBPA (100.0 µg/L) was used to study the above-mentioned extraction parameters on the SDME efficiency. All determinations were reported from the average of three replicate measurements. 3.2.1. Extraction solvent The type of organic solvent used for extracting analyte is an important factor in SDME method. The solvent should have a good affinity for the analyte, low solubility in water, be stable during the extraction process, and have good chromatographic behavior (Nagaraju & Huang, 2007). As indicated in Fig. 3, n-octanol was chosen as the best solvent for extracting analyte. It is to be noted that the 4:1 ratio (v/v) of hexane:ethylacetate, reported as the selected solvent in LLE of acrylamide, had about eight times lower efficiency than n-octanol (Areke et al., 2002; Fernandes & Soares, 2007; Pittet et al., 2004). 3.2.2. Salt addition In SDME method, the extraction of analyte can be increased or decreased by adding salt. The extraction yield can increase due to the salting out effect. However, in some cases, high salt concentrations limit the extraction since the diffusion rate of analyte into the organic phase decreases due to the changing physical properties of the solvent–water interface (Lopez-Blanco, Blanco-Cid, Cancho-Grande, & Simal-Gandara, 2003). To investigate the effect of salt addition on the extraction efficiency, different amounts of NaCl from 0 to 33% (w/v) were studied. According to the results (Fig. S1, Supplementary information), the saturated solution of NaCl (33%, w/v) yielded the best performance.
Fig. 1. Influence of the reaction temperature on the derivatization reaction yield. Concentration of acrylamide: 100.0 µg/L, sample volume: 3.0 mL, derivatization time: 15 min.
3.2.3. Extraction temperature At high temperatures, the mass transfer and diffusion of the analyte 57
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3.2.4. Extraction time Like other microextraction methods, SDME is an equilibrium–based process (Bahramifar, Yamini, Shariati-Feizabadi, & Shamsipur, 2002). To investigate the effect of time on the extraction of the target analyte, different times in the range of 0.5–50.0 min were examined. The results (Fig. S3, Supplementary information) showed that the maximum extraction was obtained in 5 min, and after that, the extraction efficiency remained constant. 3.3. Method validation To calculate the figures of merits of the method, a 3.0-mL standard solution of acrylamide was used. The optimized derivatization and extraction conditions (98 mg of (NH4)2S2O8, 46 L of HBr, derivatization time of 15.0 min at 45 °C, 1.0 µL of n-octanol, stirring rate of 300 rpm, 33% (w/v) NaCl and extraction time of 5.0 min at room temperature) were applied to determine acrylamide. The linearity of the method was in the range of 2.0–100.0 µg/L with the determination coefficient of 0.9984. The precision of the method obtained using standard solutions of the analyte at the concentration levels of 10.0, and 100.0 µg/L were 4.6 and 6.0%, respectively. The limit of detection and quantification calculated based on peak-to-peak noise (S/N = 3 and 10) were 0.60 and 2.0 µg/L, respectively. Some characteristics of the present method and other techniques used for the determination of acrylamide in food samples are listed in Table 2. The main feature of the present method, which is in the framework of green chemistry was very low consumption of extractant and derivatization reagents. Moreover, the method showed a low detection limit and short derivatization time
Fig. 3. Effect of different organic solvents on the extraction efficiency. Concentration of 2,3-dibromopropanamide: 100.0 µg/L, sample volume: 3.0 mL, solvent volume: 1.0 µL, solution temperature: 25 °C, sample pH: 7, extraction time: 10.0 min, NaCl concentration: 13.3%, w/v. Table 1 Analysis of real samples, recovery and accuracy data. Sample
Added (µg/g)
Founda (µg/g)
Recovery (%)
Potato chips
– 2.00 4.00
1.24 3.21 5.26
– 98 100
Bread
– 3.00 5.00
1.70 4.83 6.62
– 104 98
Cookie
– 4.00 8.00
1.41 5.36 9.42
– 97 99
a
3.4. Real sample analysis To evaluate the effect of matrix components on the quantification process and the method capability for analyzing real samples, the method was applied to determine acrylamide in bread, potato chips and cookie (with no creams and nuts). The standard addition method was used to quantify the analyte in the samples. The samples were spiked with the acrylamide standard solution at two concentration levels. The spiking levels were chosen based on the initial concentration of analyte in the real samples assessed by preliminary experiments. The low spiked concentration levels were 2.0, 3.0 and 4.0 µg/g for potato chips, bread, and cookie, respectively. For the high concentration level, potato chips, bread, and cookie were spiked at 4.0, 5.0 and 8.0 µg/g, respectively. There was a good linear relationship (r2 > 0.99) between the spiked amounts and the peak height of the analyte in the real samples (Fig. S4, Supplementary information). The amounts of acrylamide found in bread, potato chips and cookie were 1.7 ± 0.1, 1.24 ± 0.08
The relative standard deviations of data are between 1.5 and 9.5% (n = 3).
toward solvent drop is done faster. On the other hand, due to the increase in the solubility of organic solvent at higher temperatures and thus the decrease in the solvent volume, extraction efficiency diminishes. High temperatures can also make the drop unstable and deteriorate method repeatability (Jeannot & Cantwell, 1996). To study the effect of temperature on extraction yield, different temperatures from 15 to 50 °C were investigated. Considering the data (Fig. S2, Supplementary information), 25 °C had the best performance for analyte extraction, and consequently, the room temperature was chosen as the optimum extraction temperature.
Table 2 Analytical characteristics of different methods used for the determination of acrylamide in food samples. Method
Sample amount (g)
Derivatization reagent
Derivatization time (min)
Extraction solvent volume (mL)
LOD (µg/ L)
Reference
LLE-GC–MS
10.0
7.5 g KBr, 10.0 mL Br2, HBra
720
5
SPE-LC-MS SPE-LC-MS-MS
1.0 10.0
– –
– –
hexane:ethyl acetate (4:1, 40) H2O (4.0) + MeOH (1.0) ACN (1.0) + H2O (5.0)
23.2 10
SPE-LC-UV LLE-LC-UV LLE-GC-ECD
2.0 0.5 1.5
– 100 30
H2O (5.0) + MeOH (5.0) ethyl acetate (8.0) ethyl acetate (12.0)
30 15 0.1
SPME-GC-ECD
1.0
60
–
0.26
Yanqin Qu et al. (2013)
RP SDMEb-GC-FID SDME-GC-ECD
50.0 2.0–3.0
– 0.5 g 2-mercapto benzoic acid 1.5 g KBr, 1.0 mL KBrO3 (0.1 M) 1.0 g KBr, 1.0 mL KBrO3 (0.1 M) – 46 µL HBr, 98 mg (NH4)2S2O8
Rufianhenares and Morales (2006) Kaykhaii and Abdi (2013) Rufianhenares and Morales (2006) Oracz et al. (2011) Bermudo et al. (2006) Podgorsek et al. (2007)
– 15
H2O (3.5 × 10−3) n-octanol (1.0 × 10−3)
57 0.6
Kaykhaii and Abdi (2013) This work
a b
Acidified to pH 1–3. Reversed phase single-drop microextraction. 58
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M. Saraji, S. Javadian
Fig. 4. GC–ECD chromatograms obtained by derivatization with HBr and (NH4)2S2O8 followed by SDME of (a) unspiked potato chips and (b) potato chips spiked with 100.0 mg/L of acrylamide.
and 1.41 ± 0.11 µg/g, respectively. The spiked level and the measured amount of the analyte in the samples are listed in Table 1. As can be seen in Table 1, the spiking recovery was higher than 97%, demonstrating that the matrix components of the samples had no significant adverse effect on analyte quantification. The chromatograms of unspiked and spiked potato chips with the analyte at 4 µg/g are shown in Fig. 4.
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4. Conclusions The present SDME-GC-ECD method was successfully used to determine acrylamide in the foods. The method allowed detection of the analyte at sub-ppb concentration level. Acrylamide was simply extracted from the samples in 5.0 min, and a very small volume of the extractant (1.0 µL of n-octanol) was used. In comparison to the different derivatization methods used to determine acrylamide by GC, the toxic bromine reagent was not used in this method, and other reagents used for the derivatization of the analyte were consumed at micro amounts (46 µL of hydrobromic acid and 98 mg of ammonium peroxydisulfate). Moreover, the derivatization reaction time greatly decreased. Compared to the previously published methods for determining acrylamide in food samples, the present method as a green and environmentally friendly technique showed low detection limit, good precision, very short derivatization and extraction time, and low solvent, reagent and sample consumption. Acknowledgments The authors are grateful to the Research Council of Isfahan University of Technology (IUT, Iran) and Center of Excellence in Sensor and Green Chemistry (Iran) for the financial support of this project. Conflict of interest The authors declared no conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.foodchem.2018.08.108. References Areke, E. D. E. N. T., Ydberg, P. E. R. R., Arlsson, P. A. K., & Riksson, S. U. N. E. E. (2002). Analysis of acrylamide, a carcinogen formed in heated food stuffs. Journal of
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