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Sensitive simultaneous determination of three sulfanilamide artificial sweeters by capillary electrophoresis with on-line preconcentration and contactless conductivity detection
Food Chemistry 188 (2015) 446–451
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Sensitive simultaneous determination of three sulfanilamide artificial sweeters by capillary electrophoresis with on-line preconcentration and contactless conductivity detection Lirong Yang, ShengJi Zhou, Yuezhou Xiao, Yufeng Tang, Tianyao Xie ⇑ School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 3 October 2013 Received in revised form 30 January 2015 Accepted 17 April 2015 Available online 8 May 2015 Keywords: Capillary electrophoresis Contactless conductivity detection On-line preconcentration Acesulfame-K Sodium saccharin Sodium cyclamate
a b s t r a c t A sensitive method followed by capillary electrophoresis with on-line perconcentration and capacitively coupled contactless conductivity detection (CE–C4D) was evaluated as a novel approach for the determination of three sulfanilamide artificial sweeteners (acesulfame-K, sodium saccharin and sodium cyclamate) in beverages. The on-line preconcentration technique, namely field-amplified sample injection, coupled with CE–C4D were successfully developed and optimized. The separation was achieved within 10 min under the following conditions: an uncoated fused-silica capillary (45 cm 50 lm i.d., Leff = 40 cm), 20 mmol L 1 HAc as running buffer, separation voltage of 12 kV, electrokinetic injection of 11 kV 8 s. The detection limits of acesulfame-K, sodium saccharin and sodium cyclamate were 4.4, 6.7 and 8.8 lg L 1, respectively. The relative standard deviation varied in the range of 3.0–5.0%. Results of this study show a great potential method for the fast screening of these artificial sweeteners contents in commercial beverages. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In modern food industries, acesulfame-K, sodium saccharin and sodium cyclamate are the common artificial sweeteners and non-caloric sweeteners that have been widely used to replace sugars in foods such as soft drinks, juices, jams, candies, and many others. Those non-caloric sweeteners were mostly recommended for people that desire or need to reduce energy or sugar intakes for health reasons, as well as for individuals with diabetes (Santivarangkna, Higl, & Foerst, 2008). In the last two decades, the consumption of these low-calories foods by the worldwide population has dramatically increased. However, sodium saccharin is the oldest artificial sweetener, but it has been the center of controversy during the last few decades due to its possible carcinogenic effects. The strictest restriction is placed on its usage owing to its potential toxicity and its acceptable daily intake (ADI) value formulated by the Would Health Organization (WHO) is the lowest among these three sweeteners (FAO & WHO, 2006; Mathlouthi & Berssan, 1993), in China, 0.15–5.0 g kg 1 (GB2760-1996, 1996). Sodium cyclamate is banned in USA, while its usage is permitted in Europe (Mathlouthi & Berssan, 1993) and in China is 7–11 mg kg 1 (GB2760-1996, 1996). Acesulfame-K also has been ⇑ Corresponding author. E-mail address: [email protected] (T. Xie). http://dx.doi.org/10.1016/j.foodchem.2015.04.060 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
used in many countries. In China, the maximum amount of acesulfame-K in beverages is 300 mg kg 1 (GB2760-1996, 1996), but according to the FAO/WHO, the ADI for acesulfame-K is 15 mg kg 1 (FAO & WHO, 2006). Commercial beverages contained considerable amount of acesulfame-K, sodium saccharin and sodium cyclamate for maintaining food quality and characteristics. In order to ensure proper implementation of the existing legislation to guarantee consumer safety, sensitive and rapid analytical methods to monitor the artificial sweetener consumption are required. A number of analytical methods are available for their determination individually or simultaneously in their mixtures. These include: high-performance liquid chromatography (HPLC) (Ferrer & Thurman, 2010; Wasik, Mccourt, & Buchgraber, 2007), ion chromatography (Zhu, Guo, Ye, & James, 2005), flow injection analysis (García-Jiménez, Valencia, & Capitán-Vallvey, 2007), kinetic spectrophotometry (Ni, Xiao, & Kokot, 2009), potentiometric determination (Filho et al., 2003), and capillary electrophoresis (CE) (Bergamo, Fracassi da Silva, & de Jesus, 2011; Frazier, Inns, Dossi, Ames, & Nursten, 2000; Horie et al., 2007; Schnierle, Kappes, & Hauser, 1998) in conjunction with various detectors. Among those reports, HPLC has been the most popular choice for the determination of these artificial sweeteners. However, it encounter some disadvantages, such as the usage of toxic solvents, involvement of complex sample pre-treatments, generation of waste products, and even low sensitivity.
L. Yang et al. / Food Chemistry 188 (2015) 446–451
CE is considered as an efficient and inexpensive analytical technique, and offered an attractive alternative to traditional HPLC methods. As known, Photometric detection in the UV region is the most commonly used detection method, although for some applications it shows inadequate limits of detection because of the low UV absorptivities of the most sweeteners, especially cyclamate. Conductivity detection is a good alternative method for compounds lacking a strong UV-absorbing moiety. Using this detection technique an isotachophoresis method was published for determination of sweeteners in chewing gums and candies (Herrmannová, Krivánková, Bartos, & Vytras, 2006). Capacitively coupled contactless conductivity detection (C4D) is a relatively new approach for detection on CE. It is universal for all ionic compounds without derivatization or indirect approaches. C4D features unprecedented ease of the cell arrangement and inherent prevention of the electrode fouling (Coltro et al., 2012; Fracassi da Silva & do Lago, ˇ & Hauser, 2013; Zemann, 2003; Zemann, Schnell, 1998; Kubán Volgger, & Bonn, 1998). An earlier article (Tanyanyiwa, Abad-Villar, & Hauser, 2004) has demonstrated the possibility of using CE–C4D on a microchip platform for separation and detection of acesulfame-K and cyclamate in model solutions. Recently, Bergamo reported another CE–C4D method for the analysis of aspartame, cyclamate, saccharin and acesulfame-K in commercial soft drinks samples, and the LODs of cyclamate, saccharin and acesulfame-K were 2.5, 1.5 and 1.4 mg/L, respectively. Online preconcentration can be regarded as one of the major developments in CE specifically to overcome the sensitivity limitations (Breadmore et al., 2013; Chen, Lü, Chen, & Teng, 2012; Chien, 1991; Simpson, Quirino, & Terabe, 2008). Among these preconcentration techniques, including sweeping, stacking, focusing, dynamic pH junction and extraction, field-amplified sample injection (FASI) is the simplest method with enrichment factors of several 100-fold or higher (Chien, 1991; Zhang & Thormann, 1996). Due to its simplicity and ease of applicability, FASI is regards as a most attractive method even though the sensitivity is not the highest one. CE–C4D, coupled with on-line preconcentration technique, has been demonstrated a simple, rapid, accurate, high sensitive and cost-effective analytical method with many advantages compared with the normal CE-UV system (Coltro et al., 2012; Ji, Chen, Zhang, ˇ & Hauser, 2013). To the best of our knowlLi, & Xie, 2014; Kubán edge, no paper regarding the highly sensitive detection of three sulfanilamide artificial sweeteners (acesulfame-K, sodium saccharin and sodium cyclamate) in commercial beverages by FASI–CE– C4D is published. The aim of this work is to establish a sensitive, simple, cost effective and highly specific analytical method for simultaneous determination of acesulfame-K, sodium saccharin, and sodium cyclamate in beverages by FASI–CE–C4D. The performance of the method was evaluated with regard to the ability to generate accurate and precise qualitative and quantitative data in the relevant concentration range. These indicated its valuable potential application for the simple, sensitive analysis of trace-amount sulfanilamide artificial sweeteners in food samples.
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throughout this work. BGE was prepared fresh during the experiments. Individual standard solution (100 mg L 1) of each sweetener was prepared by dissolving the corresponding solid reagents in deionized water. Lower concentration of working standard solution used in the analysis was prepared by dilution of the respective stock solutions with deionized water, as required. 2.2. Preparation of real samples Real beverage samples were collected from the local supermarkets. Three different bands of Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03) were treated. The commercial beverage sample was degassed in an ultrasonic bath during 10 min, in order to remove the carbon dioxide gas that might be present in the beverage. Then, it was diluted with deionized water as required. The artificial sweetener in liquid form required only a dilution with deionized water before injection in the CE–C4D system. 2.3. Instrumentation and procedure The CE–C4D equipment mainly includes CES2008-C4D/CD-1B, fused-silica capillary and personal computer (Lenovo, China). The CES2008-C4D/CD-1B was manufactured by School of Chemistry and Chemical Engineering, Sun Yat-sen University, China. The C4D parameters were the same as the previous report (Wei, Li, Yang, Jiang, & Xie, 2011). The CE–C4D instrument control and data collection were performed using the computer. The fused silica capillary was obtained from Hebei Ruifeng Instrumental Co. (Handan, China). The dimension of the capillary was 50 lm i.d., 375 lm o.d., 40 cm effective length, and 45 cm total length, respectively. 2.3.1. FASI–CE–C4D procedure Before the analysis, capillary was flushed with 0.1 M NaOH, water, 0.1 M HNO3, water and the BGE for 3 min, respectively. After each run, the capillary was only rinsed with the BGE for 3 min. Unless noted, samples were injected electrokinetically with a voltage of 11.0 kV for 8 s. The separation voltage was set to
2. Materials and methods 2.1. Reagents and materials Acesulfame-K, sodium saccharin and sodium cyclamate were bought from J&K Chemical Co. (Beijing, China). Acetic acid was purchased from Guangzhou Chemical Reagent Co. (Guangzhou, China). All reagents were analytical grade unless otherwise indicated. All solutions were prepared with ultra-pure water (18 MX-cm) and stored in a refrigerator at 4 °C when not in use. 20 mmol L 1 HAc was used as the running buffer, i.e. background electrolyte (BGE),
Fig. 1. Comparison of FASI method with the conventional gravity sample injection. (A) conventional gravity injection, 5.0 mg L 1 mixture of acesulfame-K, sodium saccharin and sodium cyclamate, 20 cm 8 s; (B) FASI procedure, 0.10 mg L 1 mixture, electrokinetic injection of 11 kV 8 s. Other conditions: 20 mM HAc as the running buffer; separation voltage of 12 kV; uncoated fused-silica capillary (45 cm 50 lm i.d., Leff = 40 cm). Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate.
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Fig. 2. (A) Electropherogram of a mixture containing 0.2 lM of acesulfame-K, sodium saccharin and sodium cyclamate with FASI–CE–C4D and (B) calibration plots. Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate. Other conditions were the same as mentioned in Fig. 1.
12.0 kV. A water plug was introduced by gravity injection for 5 s at a height of 200 mm, prior to electrokinetic sample injection. At the end of each working-day, the capillary was rinsed for 10 min with water before storing it over night. To ensure good reproducibility of the analysis, all containers, vials and snap caps were ultrasonically rinsed in water, and then soaked in water for an overnight period. Limits of detection (LODs) were determined corresponding to peak height for S/N of 3. Peaks were identified by standard addition method. The experiment was done under the demanded laboratory environment with constant room temperature (25 °C) and low humidity (<60%). 2.3.2. HPLC procedure As a reference method, HPLC measurements were carried out by an Agilent 1100 series HPLC-DAD system. The procedure and working parameters were described in detail elsewhere (GB2760-1996, 1996; Lawrence & Charbonneau, 1998). All chromatograms were run at room temperature. The retention times were 10.9 min for acesulfame-K, 13.6 min for sodium cyclamate, and 16.3 min for sodium saccharin, respectively. 3. Results and discussion 3.1. Optimization of the CE separation The background electrolyte (BGE) affects the migration time and the separation between compounds directly. In the selection of BGE, the main consideration is the ionization characteristic of the analytes. The pKa values for the corresponding acids of the acesulfame-K, sodium saccharin and sodium cyclamate are 2.0, 1.8 and 1.9, respectively (Riera, Vogel, Simon, & Coutre, 2007),
which indicated that the analytes have a net negative charge in an aqueous BGE when pH > 3. Therefore, in our study, negative polarity separation voltage was used. However, in negative polarity separation voltage mode, the direction of the electroosmotic flow (EOF) was opposite to that of anion electromigration resulted in very poor resolution and detection capability when pH > 4.5, it is favorable to use the EOF modifier to suppress or reverse EOF direction. Generally, Hexadecyl Trimethyl Ammonium Bromide was used as the EOF modifier. Several electrolytes utilized as the buffer solution that possesses a useful pH range from 3.0 to 8.0 were tested, including HAc, NaAc, MES, Tris, His, and Arg. Among these tested BGE, 20 mM HAc provided satisfactory results with the highest sensitivity response relative to the others. Moreover, in such solution (pH = 3.3) the EOF is almost omitted. So EOF modifier was unnecessary and not used in the following experiments. This feature is advantageous because it can effectively avoid the problems that the obvious broadening of the analyte peaks would be observed, when attempt to carry out the same CE separation with EOF reversion. This was most likely due to interactions of the analyte with cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethyl-ammonium bromide (TTAB) that were evaluated as EOF modifiers. Tests also showed that the efficiency of the separation became good and the migration time became short, when the separation voltage was increased. As known, higher voltage will result in the peak broadening because of the Joule heating effect, the resolutions between acesulfame-K and sodium saccharin or sodium cyclamate became worse. In our study, a voltage of 12 kV was selected as the best separation voltage. 3.2. Optimization of FASI conditions In order to achieve the maximum amount of analyte loaded into the capillary with FASI, several parameters such as sample solution composition, water plug, injection voltage, and injection time must be optimized in order to obtain the best FASI efficiency. It has been showed that high enhancement in FASI can be achieved easily by dissolving the sample in a low conductivity solvent such as water or organic solvent (Zheng et al., 2008). In this study, the testing result shown that water was the best choice. Therefore, the analytes were directly dissolved in water. Several reports have certified that the reproducibility and sensitivity improved when a water plug was used in the injection (Huang, Lin, Yu, Xu, & Chen, 2008; Monton & Terabe, 2004). In our study, the gravity injection of a water plug was tested in the range of 0–10 s with 2 s increments at a height of 200 mm. As a result, an obviously increase of peak intensities was observed when the water plug was injected from 2 to 6 s. However, when a further increase of water plug was injected to 10 s, the peak intensities became low. Thus, 5 s of water plug injection was chosen. The injection voltage and injection time are the most crucial factors that affect the sensitivity enhancement in FASI. A series of injection voltages ranging from 5 to 12 kV were tested in our
Table 1 The results of linearity, limits of detection (LOD), and precision with FASI–CE–C4D method.
a b c d
Compounds
Linearity (lM)
Correlation (R, n = 6)
LODa (lM)
LOD (lg L
Aecfulfame-K Sodium saccharin Sodium cyclamate
0.05–2.6 0.06–2.6 0.07–2.6
0.998 0.997 0.996
0.022 0.032 0.044
4.4 6.7 8.8
Estimated on the basis of S/N = 3. The inter-day and intra-day analysis precision was tested at 0.20 lM levels. The intra-day analysis precision. The inter-day analysis precision.
1
)
RSD%b (n = 6) 3.6c, 4.1d 3.8, 4.2 3.9, 4.4
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Fig. 3. Electropherograms for the separation of acesulfame-K, sodium saccharin and sodium cyclamate in Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03). Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate; 4, Cl ; 5, unknown. Other conditions were the same as mentioned in Fig. 1.
Table 2 Assay results of sulfanilamide artificial sweeteners in real samples.a Sample No.
Acesulfame K (mg L 1)
Sodium saccharin (mg L 1)
Sodium cyclamate (mg L 1)
CCB01 CCB02 CCB03
214, (210)b ND, (ND) 120, (124)
ND, (ND) 125, (128) 78.1, (77.8)
ND, (ND) 273, (276) 182, (179)
ND: not detected. a Conditions are the same as in Fig. 1. b The data in the brackets are the result by the HPLC method.
study. Significant increase of peak intensity was noted when the injection voltage was increased. However, a minor peak-broadening was observed for the analytes when the injection voltage reached 11 kV. The peak broadening problem in conjunction with peak distortion became more obvious when the injection voltage increased higher. Thus, an injection voltage of 11 kV was chosen as the optimum injection voltage. Optimization of injection time was carried out ranging from 4 to 12 s with 2 s increments at 11 kV. A marked increase of peak intensity was observed when the injection time was increased from 4 to 8 s. A further increase of injection time to 12 s resulted in a serious peak broadening problem. Therefore, an injection time of 8 s was chosen as the optimum injection time.
Table 3 Recovery of spiked-standard in real samples.a
a b
In order to evaluate the sensitivity enhancement of FASI, comparison with the conventional gravity sample injection method was carried out. Fig. 1 shows the results. The injection parameters were 11 kV 8 s for FASI and 20 cm 8 s for conventional gravity sample injection, respectively. The sample concentration used to generate Fig. 1A was 5.0 mg L 1, while that for Fig. 2B was 50 times lower, i.e., 0.10 mg L 1. The peak height of Fig. 1B is much higher than that of Fig. 1A. In total, the sensitivity enhancement factor was 560, which was in accord with the expected results by FASI method (Chien, 1991; Zhang & Thormann, 1996). 3.3. Linearity, detection limits, and precision, Under the optimum FASI–CE–C4D condition, the typical electropherogram for a mixture containing 0.2 lM of acesulfame-K, sodium saccharin and sodium cyclamate was shown in Fig. 2(A), and calibration plots in Fig. 2(B). The method validation including linearity range, LODs, precision was carried out, and were summarized in Table 1. There was an excellent linearity between the peak area (mV s) and the concentration (lM) of acesulfame-K, sodium saccharin and sodium cyclamate in the range of 0.05–2.6 lM, with the correlation coefficients from 0.996 to 0.998. The LODs were 0.022 lM (4.4 lg L 1), 0.032 lM (6.7 lg L 1) and 0.044 lM (8.8 lg L 1), respectively. The inter-day and intra-day analysis precision was tested at 0.20 lM levels, RSDs were found below 5.0% (n = 6), indicating good repeatability. 3.4. Influence of other food additives
Compound
Init. amountb (mg L 1)
Added (mg L 1)
Found (mg L 1)
Recovery(%)
Acesulfame K
214 0
100 10
311 10.1
99 101
Saccharin sodium
125 0
100 10
229 9.6
101.7 96
Sodium cyclamate
273 0
100 10
369 9.67
98.9 96.7
CE conditions are the same as in Fig. 1. The initial amount of sulfanilamide artificial sweetener in original sample.
Commercial beverages not only contained amount of these three sweeteners, but also, in which other food additives such as colorants and preservatives might be present. Therefore, in this study, we examined whether preservatives (benzoic acid, salicylic acid, sorbic acid, dehydroacetic acid) and colorants (carmine, tartrazine, sunset yellow, amaranth, and brilliant blue) interfere with the detection of these three analytes in CE. As a result, the listed preservatives or colorants did not interfere the determination. Especially, benzoic acid, sorbic acid and dehydroacetic acid, as the familiar coexisted compounds in the commercial beverages,
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were not detected. Therefore, these three analytes can be analyzed with this method without any interference from possible preservatives and colorants. 3.5. Applications and recoveries Three different bands of Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03) were treated with the procedure depicted in Section 2.2 prior to FASI– CE–C4D analysis. Fig. 3 demonstrated the electropherograms. The determination results of acesulfame-K, sodium saccharin and sodium cyclamate in the samples were listed in Table 2. The results confirmed that CCB01 contain acesulfame-K only, both of sodium saccharin and sodium cyclamate were found in CCB02, and all of them were detected in CCB03, which were in accord with the label ingredients. The determination results of acesulfame-K, sodium saccharin and sodium cyclamate in real beverage samples were lower than the maximum addition levels established by China (GB2760-1996, 1996). The quantitative results were further evaluated by HPLC method (data shown in Table 2). The significance testing results revealed that the quantitative results of the proposed method were reliable. Compared with HPLC method, FASI– CE–C4D method is better for its simplicity in operation and low consumption of organic solvent. The assay indicated that the proposed analytical method shows great potential in the determination of acesulfame-K, sodium saccharin and sodium cyclamate in commercial beverages. Recovery experiments were performed by adding accurate amounts of acesulfame-K, sodium saccharin and sodium cyclamate to the real beverage samples. The standard-spiked samples were subject to the sample preparation procedure depicted in Section 2.2. The resulting recovery values were summarized in Table 3. 4. Conclusion The FASI–CE–C4D method demonstrated to be a high sensitive, simple and accurate analytical technique for the simultaneous determination of acesulfame-K, sodium saccharin and sodium cyclamate in beverage samples. By using the FASI preconcentration technique, excellent detection limits (4.4 lg L 1 for acesulfame-K, 6.7 lg L 1 for sodium saccharin and 8.8 lg L 1 for sodium cyclamate) can be achieved, these were much lower than those normal CE-UV or CE–C4D methods. This proposed method does not require complex sample preparation, but only a simple dilution or dissolution of the sample. Another advantage of this method is that no EOF modifier is required to be added into the BGE. It can effectively avoid the problems that the obvious broadening of the analyte peaks would be observed, when attempt to carry out the same CE separation with EOF reversion. This was most likely due to interactions of the analyte with CTAB and TTAB that were evaluated as EOF modifiers. This method provided a great incentive to further investigate the applicability of FASI–CE–C4D method to achieve lower detection limits than the legislated ones. Acknowledgements The authors acknowledge the financial supports from the National Science Foundation of China (NSFC, Grant No.: J1103305) and the Innovative Experiment and Research Fund for College Students of Guangdong Province. References Bergamo, A. B., Fracassi da Silva, J. A., & de Jesus, D. P. (2011). Simultaneous determination of aspartame, cyclamate, saccharin and acesulfame-K in soft drinks and tabletop sweetener formulations by capillary electrophoresis with
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Sensitive simultaneous determination of three sulfanilamide artificial sweeters by capillary electrophoresis with on-line preconcentration and contactless conductivity detection Lirong Yang, ShengJi Zhou, Yuezhou Xiao, Yufeng Tang, Tianyao Xie ⇑ School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 3 October 2013 Received in revised form 30 January 2015 Accepted 17 April 2015 Available online 8 May 2015 Keywords: Capillary electrophoresis Contactless conductivity detection On-line preconcentration Acesulfame-K Sodium saccharin Sodium cyclamate
a b s t r a c t A sensitive method followed by capillary electrophoresis with on-line perconcentration and capacitively coupled contactless conductivity detection (CE–C4D) was evaluated as a novel approach for the determination of three sulfanilamide artificial sweeteners (acesulfame-K, sodium saccharin and sodium cyclamate) in beverages. The on-line preconcentration technique, namely field-amplified sample injection, coupled with CE–C4D were successfully developed and optimized. The separation was achieved within 10 min under the following conditions: an uncoated fused-silica capillary (45 cm 50 lm i.d., Leff = 40 cm), 20 mmol L 1 HAc as running buffer, separation voltage of 12 kV, electrokinetic injection of 11 kV 8 s. The detection limits of acesulfame-K, sodium saccharin and sodium cyclamate were 4.4, 6.7 and 8.8 lg L 1, respectively. The relative standard deviation varied in the range of 3.0–5.0%. Results of this study show a great potential method for the fast screening of these artificial sweeteners contents in commercial beverages. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In modern food industries, acesulfame-K, sodium saccharin and sodium cyclamate are the common artificial sweeteners and non-caloric sweeteners that have been widely used to replace sugars in foods such as soft drinks, juices, jams, candies, and many others. Those non-caloric sweeteners were mostly recommended for people that desire or need to reduce energy or sugar intakes for health reasons, as well as for individuals with diabetes (Santivarangkna, Higl, & Foerst, 2008). In the last two decades, the consumption of these low-calories foods by the worldwide population has dramatically increased. However, sodium saccharin is the oldest artificial sweetener, but it has been the center of controversy during the last few decades due to its possible carcinogenic effects. The strictest restriction is placed on its usage owing to its potential toxicity and its acceptable daily intake (ADI) value formulated by the Would Health Organization (WHO) is the lowest among these three sweeteners (FAO & WHO, 2006; Mathlouthi & Berssan, 1993), in China, 0.15–5.0 g kg 1 (GB2760-1996, 1996). Sodium cyclamate is banned in USA, while its usage is permitted in Europe (Mathlouthi & Berssan, 1993) and in China is 7–11 mg kg 1 (GB2760-1996, 1996). Acesulfame-K also has been ⇑ Corresponding author. E-mail address: [email protected] (T. Xie). http://dx.doi.org/10.1016/j.foodchem.2015.04.060 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
used in many countries. In China, the maximum amount of acesulfame-K in beverages is 300 mg kg 1 (GB2760-1996, 1996), but according to the FAO/WHO, the ADI for acesulfame-K is 15 mg kg 1 (FAO & WHO, 2006). Commercial beverages contained considerable amount of acesulfame-K, sodium saccharin and sodium cyclamate for maintaining food quality and characteristics. In order to ensure proper implementation of the existing legislation to guarantee consumer safety, sensitive and rapid analytical methods to monitor the artificial sweetener consumption are required. A number of analytical methods are available for their determination individually or simultaneously in their mixtures. These include: high-performance liquid chromatography (HPLC) (Ferrer & Thurman, 2010; Wasik, Mccourt, & Buchgraber, 2007), ion chromatography (Zhu, Guo, Ye, & James, 2005), flow injection analysis (García-Jiménez, Valencia, & Capitán-Vallvey, 2007), kinetic spectrophotometry (Ni, Xiao, & Kokot, 2009), potentiometric determination (Filho et al., 2003), and capillary electrophoresis (CE) (Bergamo, Fracassi da Silva, & de Jesus, 2011; Frazier, Inns, Dossi, Ames, & Nursten, 2000; Horie et al., 2007; Schnierle, Kappes, & Hauser, 1998) in conjunction with various detectors. Among those reports, HPLC has been the most popular choice for the determination of these artificial sweeteners. However, it encounter some disadvantages, such as the usage of toxic solvents, involvement of complex sample pre-treatments, generation of waste products, and even low sensitivity.
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CE is considered as an efficient and inexpensive analytical technique, and offered an attractive alternative to traditional HPLC methods. As known, Photometric detection in the UV region is the most commonly used detection method, although for some applications it shows inadequate limits of detection because of the low UV absorptivities of the most sweeteners, especially cyclamate. Conductivity detection is a good alternative method for compounds lacking a strong UV-absorbing moiety. Using this detection technique an isotachophoresis method was published for determination of sweeteners in chewing gums and candies (Herrmannová, Krivánková, Bartos, & Vytras, 2006). Capacitively coupled contactless conductivity detection (C4D) is a relatively new approach for detection on CE. It is universal for all ionic compounds without derivatization or indirect approaches. C4D features unprecedented ease of the cell arrangement and inherent prevention of the electrode fouling (Coltro et al., 2012; Fracassi da Silva & do Lago, ˇ & Hauser, 2013; Zemann, 2003; Zemann, Schnell, 1998; Kubán Volgger, & Bonn, 1998). An earlier article (Tanyanyiwa, Abad-Villar, & Hauser, 2004) has demonstrated the possibility of using CE–C4D on a microchip platform for separation and detection of acesulfame-K and cyclamate in model solutions. Recently, Bergamo reported another CE–C4D method for the analysis of aspartame, cyclamate, saccharin and acesulfame-K in commercial soft drinks samples, and the LODs of cyclamate, saccharin and acesulfame-K were 2.5, 1.5 and 1.4 mg/L, respectively. Online preconcentration can be regarded as one of the major developments in CE specifically to overcome the sensitivity limitations (Breadmore et al., 2013; Chen, Lü, Chen, & Teng, 2012; Chien, 1991; Simpson, Quirino, & Terabe, 2008). Among these preconcentration techniques, including sweeping, stacking, focusing, dynamic pH junction and extraction, field-amplified sample injection (FASI) is the simplest method with enrichment factors of several 100-fold or higher (Chien, 1991; Zhang & Thormann, 1996). Due to its simplicity and ease of applicability, FASI is regards as a most attractive method even though the sensitivity is not the highest one. CE–C4D, coupled with on-line preconcentration technique, has been demonstrated a simple, rapid, accurate, high sensitive and cost-effective analytical method with many advantages compared with the normal CE-UV system (Coltro et al., 2012; Ji, Chen, Zhang, ˇ & Hauser, 2013). To the best of our knowlLi, & Xie, 2014; Kubán edge, no paper regarding the highly sensitive detection of three sulfanilamide artificial sweeteners (acesulfame-K, sodium saccharin and sodium cyclamate) in commercial beverages by FASI–CE– C4D is published. The aim of this work is to establish a sensitive, simple, cost effective and highly specific analytical method for simultaneous determination of acesulfame-K, sodium saccharin, and sodium cyclamate in beverages by FASI–CE–C4D. The performance of the method was evaluated with regard to the ability to generate accurate and precise qualitative and quantitative data in the relevant concentration range. These indicated its valuable potential application for the simple, sensitive analysis of trace-amount sulfanilamide artificial sweeteners in food samples.
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throughout this work. BGE was prepared fresh during the experiments. Individual standard solution (100 mg L 1) of each sweetener was prepared by dissolving the corresponding solid reagents in deionized water. Lower concentration of working standard solution used in the analysis was prepared by dilution of the respective stock solutions with deionized water, as required. 2.2. Preparation of real samples Real beverage samples were collected from the local supermarkets. Three different bands of Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03) were treated. The commercial beverage sample was degassed in an ultrasonic bath during 10 min, in order to remove the carbon dioxide gas that might be present in the beverage. Then, it was diluted with deionized water as required. The artificial sweetener in liquid form required only a dilution with deionized water before injection in the CE–C4D system. 2.3. Instrumentation and procedure The CE–C4D equipment mainly includes CES2008-C4D/CD-1B, fused-silica capillary and personal computer (Lenovo, China). The CES2008-C4D/CD-1B was manufactured by School of Chemistry and Chemical Engineering, Sun Yat-sen University, China. The C4D parameters were the same as the previous report (Wei, Li, Yang, Jiang, & Xie, 2011). The CE–C4D instrument control and data collection were performed using the computer. The fused silica capillary was obtained from Hebei Ruifeng Instrumental Co. (Handan, China). The dimension of the capillary was 50 lm i.d., 375 lm o.d., 40 cm effective length, and 45 cm total length, respectively. 2.3.1. FASI–CE–C4D procedure Before the analysis, capillary was flushed with 0.1 M NaOH, water, 0.1 M HNO3, water and the BGE for 3 min, respectively. After each run, the capillary was only rinsed with the BGE for 3 min. Unless noted, samples were injected electrokinetically with a voltage of 11.0 kV for 8 s. The separation voltage was set to
2. Materials and methods 2.1. Reagents and materials Acesulfame-K, sodium saccharin and sodium cyclamate were bought from J&K Chemical Co. (Beijing, China). Acetic acid was purchased from Guangzhou Chemical Reagent Co. (Guangzhou, China). All reagents were analytical grade unless otherwise indicated. All solutions were prepared with ultra-pure water (18 MX-cm) and stored in a refrigerator at 4 °C when not in use. 20 mmol L 1 HAc was used as the running buffer, i.e. background electrolyte (BGE),
Fig. 1. Comparison of FASI method with the conventional gravity sample injection. (A) conventional gravity injection, 5.0 mg L 1 mixture of acesulfame-K, sodium saccharin and sodium cyclamate, 20 cm 8 s; (B) FASI procedure, 0.10 mg L 1 mixture, electrokinetic injection of 11 kV 8 s. Other conditions: 20 mM HAc as the running buffer; separation voltage of 12 kV; uncoated fused-silica capillary (45 cm 50 lm i.d., Leff = 40 cm). Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate.
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Fig. 2. (A) Electropherogram of a mixture containing 0.2 lM of acesulfame-K, sodium saccharin and sodium cyclamate with FASI–CE–C4D and (B) calibration plots. Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate. Other conditions were the same as mentioned in Fig. 1.
12.0 kV. A water plug was introduced by gravity injection for 5 s at a height of 200 mm, prior to electrokinetic sample injection. At the end of each working-day, the capillary was rinsed for 10 min with water before storing it over night. To ensure good reproducibility of the analysis, all containers, vials and snap caps were ultrasonically rinsed in water, and then soaked in water for an overnight period. Limits of detection (LODs) were determined corresponding to peak height for S/N of 3. Peaks were identified by standard addition method. The experiment was done under the demanded laboratory environment with constant room temperature (25 °C) and low humidity (<60%). 2.3.2. HPLC procedure As a reference method, HPLC measurements were carried out by an Agilent 1100 series HPLC-DAD system. The procedure and working parameters were described in detail elsewhere (GB2760-1996, 1996; Lawrence & Charbonneau, 1998). All chromatograms were run at room temperature. The retention times were 10.9 min for acesulfame-K, 13.6 min for sodium cyclamate, and 16.3 min for sodium saccharin, respectively. 3. Results and discussion 3.1. Optimization of the CE separation The background electrolyte (BGE) affects the migration time and the separation between compounds directly. In the selection of BGE, the main consideration is the ionization characteristic of the analytes. The pKa values for the corresponding acids of the acesulfame-K, sodium saccharin and sodium cyclamate are 2.0, 1.8 and 1.9, respectively (Riera, Vogel, Simon, & Coutre, 2007),
which indicated that the analytes have a net negative charge in an aqueous BGE when pH > 3. Therefore, in our study, negative polarity separation voltage was used. However, in negative polarity separation voltage mode, the direction of the electroosmotic flow (EOF) was opposite to that of anion electromigration resulted in very poor resolution and detection capability when pH > 4.5, it is favorable to use the EOF modifier to suppress or reverse EOF direction. Generally, Hexadecyl Trimethyl Ammonium Bromide was used as the EOF modifier. Several electrolytes utilized as the buffer solution that possesses a useful pH range from 3.0 to 8.0 were tested, including HAc, NaAc, MES, Tris, His, and Arg. Among these tested BGE, 20 mM HAc provided satisfactory results with the highest sensitivity response relative to the others. Moreover, in such solution (pH = 3.3) the EOF is almost omitted. So EOF modifier was unnecessary and not used in the following experiments. This feature is advantageous because it can effectively avoid the problems that the obvious broadening of the analyte peaks would be observed, when attempt to carry out the same CE separation with EOF reversion. This was most likely due to interactions of the analyte with cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethyl-ammonium bromide (TTAB) that were evaluated as EOF modifiers. Tests also showed that the efficiency of the separation became good and the migration time became short, when the separation voltage was increased. As known, higher voltage will result in the peak broadening because of the Joule heating effect, the resolutions between acesulfame-K and sodium saccharin or sodium cyclamate became worse. In our study, a voltage of 12 kV was selected as the best separation voltage. 3.2. Optimization of FASI conditions In order to achieve the maximum amount of analyte loaded into the capillary with FASI, several parameters such as sample solution composition, water plug, injection voltage, and injection time must be optimized in order to obtain the best FASI efficiency. It has been showed that high enhancement in FASI can be achieved easily by dissolving the sample in a low conductivity solvent such as water or organic solvent (Zheng et al., 2008). In this study, the testing result shown that water was the best choice. Therefore, the analytes were directly dissolved in water. Several reports have certified that the reproducibility and sensitivity improved when a water plug was used in the injection (Huang, Lin, Yu, Xu, & Chen, 2008; Monton & Terabe, 2004). In our study, the gravity injection of a water plug was tested in the range of 0–10 s with 2 s increments at a height of 200 mm. As a result, an obviously increase of peak intensities was observed when the water plug was injected from 2 to 6 s. However, when a further increase of water plug was injected to 10 s, the peak intensities became low. Thus, 5 s of water plug injection was chosen. The injection voltage and injection time are the most crucial factors that affect the sensitivity enhancement in FASI. A series of injection voltages ranging from 5 to 12 kV were tested in our
Table 1 The results of linearity, limits of detection (LOD), and precision with FASI–CE–C4D method.
a b c d
Compounds
Linearity (lM)
Correlation (R, n = 6)
LODa (lM)
LOD (lg L
Aecfulfame-K Sodium saccharin Sodium cyclamate
0.05–2.6 0.06–2.6 0.07–2.6
0.998 0.997 0.996
0.022 0.032 0.044
4.4 6.7 8.8
Estimated on the basis of S/N = 3. The inter-day and intra-day analysis precision was tested at 0.20 lM levels. The intra-day analysis precision. The inter-day analysis precision.
1
)
RSD%b (n = 6) 3.6c, 4.1d 3.8, 4.2 3.9, 4.4
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Fig. 3. Electropherograms for the separation of acesulfame-K, sodium saccharin and sodium cyclamate in Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03). Peak identification: 1, acesulfame-K; 2, sodium saccharin; 3, sodium cyclamate; 4, Cl ; 5, unknown. Other conditions were the same as mentioned in Fig. 1.
Table 2 Assay results of sulfanilamide artificial sweeteners in real samples.a Sample No.
Acesulfame K (mg L 1)
Sodium saccharin (mg L 1)
Sodium cyclamate (mg L 1)
CCB01 CCB02 CCB03
214, (210)b ND, (ND) 120, (124)
ND, (ND) 125, (128) 78.1, (77.8)
ND, (ND) 273, (276) 182, (179)
ND: not detected. a Conditions are the same as in Fig. 1. b The data in the brackets are the result by the HPLC method.
study. Significant increase of peak intensity was noted when the injection voltage was increased. However, a minor peak-broadening was observed for the analytes when the injection voltage reached 11 kV. The peak broadening problem in conjunction with peak distortion became more obvious when the injection voltage increased higher. Thus, an injection voltage of 11 kV was chosen as the optimum injection voltage. Optimization of injection time was carried out ranging from 4 to 12 s with 2 s increments at 11 kV. A marked increase of peak intensity was observed when the injection time was increased from 4 to 8 s. A further increase of injection time to 12 s resulted in a serious peak broadening problem. Therefore, an injection time of 8 s was chosen as the optimum injection time.
Table 3 Recovery of spiked-standard in real samples.a
a b
In order to evaluate the sensitivity enhancement of FASI, comparison with the conventional gravity sample injection method was carried out. Fig. 1 shows the results. The injection parameters were 11 kV 8 s for FASI and 20 cm 8 s for conventional gravity sample injection, respectively. The sample concentration used to generate Fig. 1A was 5.0 mg L 1, while that for Fig. 2B was 50 times lower, i.e., 0.10 mg L 1. The peak height of Fig. 1B is much higher than that of Fig. 1A. In total, the sensitivity enhancement factor was 560, which was in accord with the expected results by FASI method (Chien, 1991; Zhang & Thormann, 1996). 3.3. Linearity, detection limits, and precision, Under the optimum FASI–CE–C4D condition, the typical electropherogram for a mixture containing 0.2 lM of acesulfame-K, sodium saccharin and sodium cyclamate was shown in Fig. 2(A), and calibration plots in Fig. 2(B). The method validation including linearity range, LODs, precision was carried out, and were summarized in Table 1. There was an excellent linearity between the peak area (mV s) and the concentration (lM) of acesulfame-K, sodium saccharin and sodium cyclamate in the range of 0.05–2.6 lM, with the correlation coefficients from 0.996 to 0.998. The LODs were 0.022 lM (4.4 lg L 1), 0.032 lM (6.7 lg L 1) and 0.044 lM (8.8 lg L 1), respectively. The inter-day and intra-day analysis precision was tested at 0.20 lM levels, RSDs were found below 5.0% (n = 6), indicating good repeatability. 3.4. Influence of other food additives
Compound
Init. amountb (mg L 1)
Added (mg L 1)
Found (mg L 1)
Recovery(%)
Acesulfame K
214 0
100 10
311 10.1
99 101
Saccharin sodium
125 0
100 10
229 9.6
101.7 96
Sodium cyclamate
273 0
100 10
369 9.67
98.9 96.7
CE conditions are the same as in Fig. 1. The initial amount of sulfanilamide artificial sweetener in original sample.
Commercial beverages not only contained amount of these three sweeteners, but also, in which other food additives such as colorants and preservatives might be present. Therefore, in this study, we examined whether preservatives (benzoic acid, salicylic acid, sorbic acid, dehydroacetic acid) and colorants (carmine, tartrazine, sunset yellow, amaranth, and brilliant blue) interfere with the detection of these three analytes in CE. As a result, the listed preservatives or colorants did not interfere the determination. Especially, benzoic acid, sorbic acid and dehydroacetic acid, as the familiar coexisted compounds in the commercial beverages,
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were not detected. Therefore, these three analytes can be analyzed with this method without any interference from possible preservatives and colorants. 3.5. Applications and recoveries Three different bands of Chinese chrysanthemum beverage samples with the batch number (CCB01, CCB02, and CCB03) were treated with the procedure depicted in Section 2.2 prior to FASI– CE–C4D analysis. Fig. 3 demonstrated the electropherograms. The determination results of acesulfame-K, sodium saccharin and sodium cyclamate in the samples were listed in Table 2. The results confirmed that CCB01 contain acesulfame-K only, both of sodium saccharin and sodium cyclamate were found in CCB02, and all of them were detected in CCB03, which were in accord with the label ingredients. The determination results of acesulfame-K, sodium saccharin and sodium cyclamate in real beverage samples were lower than the maximum addition levels established by China (GB2760-1996, 1996). The quantitative results were further evaluated by HPLC method (data shown in Table 2). The significance testing results revealed that the quantitative results of the proposed method were reliable. Compared with HPLC method, FASI– CE–C4D method is better for its simplicity in operation and low consumption of organic solvent. The assay indicated that the proposed analytical method shows great potential in the determination of acesulfame-K, sodium saccharin and sodium cyclamate in commercial beverages. Recovery experiments were performed by adding accurate amounts of acesulfame-K, sodium saccharin and sodium cyclamate to the real beverage samples. The standard-spiked samples were subject to the sample preparation procedure depicted in Section 2.2. The resulting recovery values were summarized in Table 3. 4. Conclusion The FASI–CE–C4D method demonstrated to be a high sensitive, simple and accurate analytical technique for the simultaneous determination of acesulfame-K, sodium saccharin and sodium cyclamate in beverage samples. By using the FASI preconcentration technique, excellent detection limits (4.4 lg L 1 for acesulfame-K, 6.7 lg L 1 for sodium saccharin and 8.8 lg L 1 for sodium cyclamate) can be achieved, these were much lower than those normal CE-UV or CE–C4D methods. This proposed method does not require complex sample preparation, but only a simple dilution or dissolution of the sample. Another advantage of this method is that no EOF modifier is required to be added into the BGE. It can effectively avoid the problems that the obvious broadening of the analyte peaks would be observed, when attempt to carry out the same CE separation with EOF reversion. This was most likely due to interactions of the analyte with CTAB and TTAB that were evaluated as EOF modifiers. This method provided a great incentive to further investigate the applicability of FASI–CE–C4D method to achieve lower detection limits than the legislated ones. Acknowledgements The authors acknowledge the financial supports from the National Science Foundation of China (NSFC, Grant No.: J1103305) and the Innovative Experiment and Research Fund for College Students of Guangdong Province. References Bergamo, A. B., Fracassi da Silva, J. A., & de Jesus, D. P. (2011). Simultaneous determination of aspartame, cyclamate, saccharin and acesulfame-K in soft drinks and tabletop sweetener formulations by capillary electrophoresis with
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