Food Chemistry 183 (2015) 26–29
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
An environment-friendly procedure for the high performance liquid chromatography determination of benzoic acid and sorbic acid in soy sauce Mingzhen Ding ⇑, Jing Peng, Shaoling Ma, Yuchao Zhang Jinling College, Nanjing University, Nanjing 210089, China
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 6 February 2015 Accepted 9 March 2015 Available online 14 March 2015 Keywords: Environment-friendly procedure HPLC Benzoic acid Sorbic acid Internal standard method Soy sauce
a b s t r a c t A rapid, accurate and environment-friendly procedure has been developed for the HPLC-based determination of benzoic acid and sorbic acid contents in soy sauce. A C18 column served as the stationary phase, methanol–ammonium acetate buffer (0.02 M) (30:70, v/v) was used as the mobile phase, the flow rate was 1 mL/min, the UV detector was set at 225 nm and cinnamic acid was selected as an internal standard. Under such optimized conditions, benzoic acid, sorbic acid and the internal standard were separated within 8.1 min. This newly developed procedure also showed excellent recurrence, with a relative standard deviation of less than 3% and recoveries were 96.1–104.3%. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Soy sauce is a condiment that originated in ancient China and is extensively used in modern China and Southeast Asia. Benzoic acid and sorbic acid are prevalently used as preservatives in soy sauce. However, adverse effects have been associated with benzoic acid at low doses. In sensitive individuals, conditions such as asthma, urticaria, metabolic acidosis and convulsions have been observed (Tfouni & Toledo, 2002; WHO, 2000). Some clastogenic activity has also been observed with in vitro assays, therefore, the use of benzoic acid must be controlled. Sorbic acid and its leopoldite are safe and effective preservatives recommended by the Food and Agriculture Organization (FAO) and World Health Organization (WHO). However, strict standards have been set by the China Health Ministry to monitor its usage, which have been implemented since 2004. The maximum allowed combined content of benzoic acid and sorbic acid in soy sauce is 1.0 g/kg (GB/T 5009.29, 2003). Therefore, it is necessary to ensure that low levels of these preservatives are used in soy sauce to meet regulatory standards. Many methods for the determination of benzoic and sorbic acids in soy sauce are available in the literature. HPLC is the most
⇑ Corresponding author. Tel.: +86 18625186226; fax: +86 25 58646637. E-mail address: [email protected] (M. Ding). http://dx.doi.org/10.1016/j.foodchem.2015.03.025 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
popular method for their simultaneous determination (Kritsunankul & Jakmunee, 2011; Nour, Trandafir, & Ionica, 2009; Tfouni & Toledo, 2002; Wu, He, & Guo, 2012). In addition, gas chromatography (Jurado-Sanchez, Ballesteros, & Gallego, 2011; Sun, Wang, Huang, Pan, & Wang, 2013), capillary electrophoresis (Hsu, Hu, & Chiu, 2014; Wei, Li, Yang, Jiang, & Xie, 2011) and supercritical fluid chromatography (Berger & Berger, 2013) have also been utilized. However, the extensive use of organic solvents in sample preparation or in the preparation of standard solutions of the preservatives is potentially harmful to environment and the operators. In this study, we developed an HPLC-based method using small amounts of organic solvents in the liquid–liquid extraction (diethyl ether, 5 mL) and using no organic solvents in the preparation of the standard solutions. Meanwhile, benzoic and sorbic acids were determined by the internal standard method which eliminated target losses during liquid–liquid extraction, thus making the procedure more accurate.
2. Experimental 2.1. Materials and reagents Different brands of soy sauce samples were purchased from local supermarkets. Standards of benzoic acid (BA, 99.5%), sorbic acid (SA, 99%) and cinnamic acid (CA, 99%) were purchased from
M. Ding et al. / Food Chemistry 183 (2015) 26–29
Fluka (Shanghai, China). Sodium hydroxide (99%), ammonium acetate (98%) and hydrochloric acid (12 mol/L) were obtained from Hewei Chemical Co. (Shanghai, China). Methanol (HPLC grade) was purchased from Zhengxin Chemical Co. (Shanghai, China).
2.2. Preparation and storage of standards Standard stock solutions containing 1000 mg/L each of BA or SA was prepared in water (Milli-Q). The mixture was shaken well until a homogenous and clear solution formed. If necessary, a small amount of methanol was added to help dissolution. The same method was applied to prepare a 500 mg/L internal standard CA stock solution. All stock solutions were covered with aluminum foil, stored in a freezer (4 °C) and away from light (ready for a maximum usage of one month). Before each experiment, BA and SA standard solutions were prepared in the same volumetric flask, and then diluted to five different concentrations in the range 20– 100 mg/L at constant intervals, where 50 mg/L internal standard (CA) was added to every diluted standard solution.
2.3. Sample preparation 2.3.1. Extraction procedure Approximately 1 g of sample was accurately weighed in a 10 mL capped test tube. A 0.5 mL aliquot of the internal standard CA stock solution, 0.5 mL hydrochloric acid–water (1:1, v/v) and 5 mL diethyl ether were then added successively. The mixture was then sonicated for 1 min before being allowed to separate into layers. The upper ether layer was quantitatively transferred by an injector to another 10 mL capped test tube.
2.3.2. Back extraction procedure A 1 mL aliquot of aqueous sodium hydroxide (0.1 M) was added to the diethyl ether extracts. The resulting mixture was sonicated for 1 min, and then allowed to separate into layers. The upper ether layer was discarded, and the lower aqueous layer was placed in a 40 °C water bath to evaporate any residual diethyl ether. Finally, the residue was transferred to a 5 mL volumetric flask and diluted with water to scale.
27
3.2. Optimization of analysis conditions In order to obtain maximum sensitivity, it is optimal to detect each compound at its maximum absorbance wavelength. The UV–vis spectra of the compounds are shown in Fig. 1. It can be anticipated that a small peak would be obtained for BA if the detection wavelength was fixed at 275 nm. On the other hand, small peaks for BA and CA would be obtained if the detection wavelength was set at 235 nm. The maximum peak for BA could be obtained if the detector was set at 225 nm where peak intensities for BA, SA and CA were the same. Therefore, 225 nm was selected as a suitable detection wavelength. In order to verify this prediction, a standard mixture of BA, SA and CA was injected into the HPLC column at maximum absorption wavelengths of 235 and 225 nm separately. As shown in Fig. 2(a), it is evident that the peaks of BA and CA were too small when the detector was set at 235 nm. However, in Fig. 2(b), all peaks were of satisfactory intensity when the detector was set at 225 nm. To protect the environment and the health of the operators, the use of organic solvents (especially highly toxic solvents such as methanol), should be reduced as much as possible. It is generally acknowledged that to minimize matrix effects, the solvent for preparation of samples and standard solutions should be identical. In this work, the extraction-stripping two-step procedure in the sample preparation step eventually drove the sample into water matrix. This allows water to be used for preparing the BA and SA standard solutions, thus decreasing the quantity of organic solvent. To select a suitable internal standard, an extensive literature survey was done. However, no suitable internal standard has been reported so far, for the determination of BA and SA content by HPLC. Phthalate, hendecanoic acid and decylic acid are common internal standards for the determination of BA and SA content in soy sauce by gas chromatography (in China). However, these internal standards were not suitable for the technique in this study. Phthalate could get hydrolyzed by aqueous sodium hydroxide in the back extraction process. Furthermore, hendecanoic acid and decylic acid have poor UV absorption in the 190–400 nm range and are unsuitable for HPLC with an UV detector. Cinnamic acid was selected for this study due to its similar structure with benzoic acid. The initial results were promising, so cinnamic acid was chosen as the internal standard in this new developed method.
2.4. Chromatographic conditions Analytical separation was carried out on an Agilent 1100 HPLC unit using a Supelco 516 C18 column (25 cm 4.6 mm, 5 lm) at room temperature. The detector used was an UV–vis spectrophotometer set at 225 nm and the volume of sample injected was 20 lL. The mobile phase used was methanol–ammonium acetate buffer (0.02 M) (30:70, v/v); the flow rate was 1 mL/min.
3. Results and discussion 3.1. Calibration curve and detection limit The calibration curve was constructed by plotting the peak area ratio of BA or SA and the internal standard CA against the concentration of BA or SA. Excellent linearity was obtained within the concentration range of 20–100 mg/L (the wavelength used to perform the calibration was 225 nm), giving both samples a correlation coefficient of 0.9996. The detection limit of BA, SA and CA was found to be 0.2, 0.1 and 0.5 mg/L respectively in the final solution when the signal-to-noise ratio was greater than 3:1.
Fig. 1. UV spectra of BA, SA and CA (10 mg/L each); the solvent was methanol– ammonium acetate buffer (0.02 M) (30:70, v/v).
28
M. Ding et al. / Food Chemistry 183 (2015) 26–29
Fig. 2. Typical chromatogram of a standard mixture of BA, SA and CA. mg/L: BA, 40; SA, 40; CA, 50. The detector wavelength was set at: (a) 235 nm; (b) 225 nm.
Table 2 BA and SA contents in commercial soy sauce analyzed by HPLC. Analyte Soy Soy Soy Soy b
sauce sauce sauce sauce
1 2 3 4
BA (lg/g) ± SD
SA (lg/g) ± SD
238.7 ± 2.9 336.1 ± 2.7 661.3 ± 5.7 292.8 ± 2.0
246.4 ± 3.1 NDb ND 311.7 ± 2.9
ND, not detectable; SD, standard deviation; n = 3.
away from light). It was found that the relative standard deviation (RSD) values obtained were less than 3%. There was no significant influence of the stability of the preservatives on the experimental results during sample preparation and HPLC analysis. The accuracy of the current method was validated through recovery studies of the preservatives. BA and SA with two concentration levels were added to real samples of different soy sauces, and the resulting mixtures were analyzed by the method developed. The technique provided recovery percentage values from 96.1% to 104.3%, as shown in Table 1.
Fig. 3. Chromatogram of the soy sauce extraction.
3.3. Chromatogram of soy sauce extraction The chromatogram of soy sauce extraction is shown in Fig. 3. As shown in Fig. 3, separation of all the components was achieved in less than 8.1 min. The retention times of BA, SA and CA were 3.654, 4.497 and 8.007 min, respectively.
3.5. Analysis of commercial soy sauce Commercial soy sauce samples were analyzed by the developed procedure. As shown in Table 2, none of the positive samples violate the legal limit of 1.0 g/kg for BA or SA, either independently or in combination.
3.4. Precision and recovery studies 3.6. Compared with the standard method The repeatability of the method was judged by triplicate HPLC analysis with the same soy sauce under the same sample preparation procedure. The reproducibility over different days was tested for the determination of the same standard mixtures, which were stored in a refrigerator and covered by aluminum foil (to keep
To investigate the accuracy of the developed method, the National Standards of the People’s Republic of China (GB/T 23495, 2009) was employed to determine the BA and SA contents in soy sauce (Table 3). As shown in Table 3, the quantitative results
Table 1 Recovery of BA and SA from spiked soy sauce samples analyzed by HPLC.
a
Analyte
Previous (lg/g) ± SDa BA
SA
BA
SA
BA
SA
BA
SA
Soy sauce 1
238.7 ± 2.9
246.4 ± 3.1
Soy sauce 2
336.1 ± 2.7
0
100 200 100 200
100 200 100 200
337.3 ± 2.9 430.9 ± 3.6 434.2 ± 4.9 531.7 ± 4.1
346.7 ± 2.1 444.2 ± 3.8 104.3 ± 2.5 202.6 ± 3.4
98.6 96.1 98.1 97.8
100.3 98.9 104.3 101.3
SD, standard deviation; n = 3.
Added (lg)
Found (lg /g) ± SD
Recovery (%)
M. Ding et al. / Food Chemistry 183 (2015) 26–29 Table 3 Comparison of BA and SA contents found in commercial soy sauce. Analyte
The method developed
Standard method
BA (lg/g) ± SD SA (lg/g) ± SD BA (lg/g) ± SD SA (lg/g) ± SD Soy sauce 1 238.7 ± 2.9 Soy sauce 2 336.1 ± 2.7 Soy sauce 3 661.3 ± 5.7
246.4 ± 3.1 ND ND
241.8 ± 2.2 339.1 ± 3.4 663.3 ± 4.9
246.5 ± 2.6 ND ND
29
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 03.025.
References
SD, standard deviation; n = 3; ND, not detectable.
of three different soy sauce samples are in good agreement with those measured by the standard method. 4. Conclusions A rapid, accurate and environment-friendly procedure was developed for the quantitative determination of BA and SA in soy sauce. Under the recommended conditions, separation of all the components was achieved in less than 8.1 min. Sample preparation includes a two-step procedure: extraction and back extraction. The total amount of organic solvent used was only 5 mL of diethyl ether in sample preparation, and no organic solvent was used in the preparation of the standard solutions. Meanwhile, benzoic and sorbic acids were determined by an internal standard method, which greatly reduced the effects of target losses in extraction and back extraction. Thus the experimental results were quite accurate with the relative standard deviations (RSD) of less than 3% and the recoveries were 96.1–104.3%. These advantages make this method a potential and valuable tool to determine BA and SA quantitatively in soy sauce, which could be used for industrial purposes. Acknowledgement This project was financially supported by Natural Science Foundation of Jiangsu Province of China (BK 20131343 and BK 20141391).
Berger, T. A., & Berger, B. K. (2013). Rapid, direct quantitation of the preservatives benzoic and sorbic acid (and salts) plus caffeine in foods and aqueous beverages using supercritical fluid chromatography. Chromatographia, 76, 393–399. GB/T 23495. 2009. Determination of benzoic acid, sorbic acid and saccharin sodium in foods – High performance liquid chromatography method. GB/T 5009.29. 2003. Determination of sorbic acid and benzoic acid in foods. Hsu, S. H., Hu, C. C., & Chiu, T. C. (2014). Online dynamic pH junction-sweeping for the determination of benzoic and sorbic acids in food products by capillary electrophoresis. Analytical and Bioanalytical Chemistry, 406(2), 635–641. Jurado-Sanchez, B., Ballesteros, E., & Gallego, M. (2011). Gas chromatographic determination of 29 organic acids in foodstuffs after continuous solid-phase extraction. Talanta, 84(3), 924–930. Kritsunankul, O., & Jakmunee, J. (2011). Simultaneous determination of some food additives in soft drinks and other liquid foods by flow injection on-line dialysis coupled to high performance liquid chromatography. Talanta, 84(5), 1342–1349. Nour, V., Trandafir, I., & Ionica, M. E. (2009). Simultaneous determination of sorbic and benzoic acids in tomato sauce and ketchup using high-performance liquid chromatography. Annals: Food Science and Technology, 10(1), 157–162. Sun, Y., Wang, X., Huang, Y., Pan, Z., & Wang, L. (2013). Derivatization following hollow-fiber microextraction with tetramethylammonium acetate as a dualfunction regent for the determination of benzoic acid and sorbic acid by GC. Journal of Separation Science, 36(14), 2268–2276. Tfouni, S. A. V., & Toledo, M. C. F. (2002). Determination of benzoic and sorbic acids in Brazilian food. Food Control, 13(2), 117–123. Wei, R., Li, W., Yang, L., Jiang, Y., & Xie, T. (2011). Online preconcentration in capillary electrophoresis with contactless conductivity detection for sensitive determination of sorbic and benzoic acids in soy sauce. Talanta, 83(5), 1487–1490. WHO, 2000. Benzoic acid and sodium benzoate. Concise International Chemical Assessment Document, 26. Wu, Z., He, Z., & Guo, M. (2012). Determination of nine food additives in condiment by high performance liquid chromatography. Applied Mechanics and Materials, 140, 302–308.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
An environment-friendly procedure for the high performance liquid chromatography determination of benzoic acid and sorbic acid in soy sauce Mingzhen Ding ⇑, Jing Peng, Shaoling Ma, Yuchao Zhang Jinling College, Nanjing University, Nanjing 210089, China
a r t i c l e
i n f o
Article history: Received 7 November 2014 Received in revised form 6 February 2015 Accepted 9 March 2015 Available online 14 March 2015 Keywords: Environment-friendly procedure HPLC Benzoic acid Sorbic acid Internal standard method Soy sauce
a b s t r a c t A rapid, accurate and environment-friendly procedure has been developed for the HPLC-based determination of benzoic acid and sorbic acid contents in soy sauce. A C18 column served as the stationary phase, methanol–ammonium acetate buffer (0.02 M) (30:70, v/v) was used as the mobile phase, the flow rate was 1 mL/min, the UV detector was set at 225 nm and cinnamic acid was selected as an internal standard. Under such optimized conditions, benzoic acid, sorbic acid and the internal standard were separated within 8.1 min. This newly developed procedure also showed excellent recurrence, with a relative standard deviation of less than 3% and recoveries were 96.1–104.3%. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Soy sauce is a condiment that originated in ancient China and is extensively used in modern China and Southeast Asia. Benzoic acid and sorbic acid are prevalently used as preservatives in soy sauce. However, adverse effects have been associated with benzoic acid at low doses. In sensitive individuals, conditions such as asthma, urticaria, metabolic acidosis and convulsions have been observed (Tfouni & Toledo, 2002; WHO, 2000). Some clastogenic activity has also been observed with in vitro assays, therefore, the use of benzoic acid must be controlled. Sorbic acid and its leopoldite are safe and effective preservatives recommended by the Food and Agriculture Organization (FAO) and World Health Organization (WHO). However, strict standards have been set by the China Health Ministry to monitor its usage, which have been implemented since 2004. The maximum allowed combined content of benzoic acid and sorbic acid in soy sauce is 1.0 g/kg (GB/T 5009.29, 2003). Therefore, it is necessary to ensure that low levels of these preservatives are used in soy sauce to meet regulatory standards. Many methods for the determination of benzoic and sorbic acids in soy sauce are available in the literature. HPLC is the most
⇑ Corresponding author. Tel.: +86 18625186226; fax: +86 25 58646637. E-mail address: [email protected] (M. Ding). http://dx.doi.org/10.1016/j.foodchem.2015.03.025 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
popular method for their simultaneous determination (Kritsunankul & Jakmunee, 2011; Nour, Trandafir, & Ionica, 2009; Tfouni & Toledo, 2002; Wu, He, & Guo, 2012). In addition, gas chromatography (Jurado-Sanchez, Ballesteros, & Gallego, 2011; Sun, Wang, Huang, Pan, & Wang, 2013), capillary electrophoresis (Hsu, Hu, & Chiu, 2014; Wei, Li, Yang, Jiang, & Xie, 2011) and supercritical fluid chromatography (Berger & Berger, 2013) have also been utilized. However, the extensive use of organic solvents in sample preparation or in the preparation of standard solutions of the preservatives is potentially harmful to environment and the operators. In this study, we developed an HPLC-based method using small amounts of organic solvents in the liquid–liquid extraction (diethyl ether, 5 mL) and using no organic solvents in the preparation of the standard solutions. Meanwhile, benzoic and sorbic acids were determined by the internal standard method which eliminated target losses during liquid–liquid extraction, thus making the procedure more accurate.
2. Experimental 2.1. Materials and reagents Different brands of soy sauce samples were purchased from local supermarkets. Standards of benzoic acid (BA, 99.5%), sorbic acid (SA, 99%) and cinnamic acid (CA, 99%) were purchased from
M. Ding et al. / Food Chemistry 183 (2015) 26–29
Fluka (Shanghai, China). Sodium hydroxide (99%), ammonium acetate (98%) and hydrochloric acid (12 mol/L) were obtained from Hewei Chemical Co. (Shanghai, China). Methanol (HPLC grade) was purchased from Zhengxin Chemical Co. (Shanghai, China).
2.2. Preparation and storage of standards Standard stock solutions containing 1000 mg/L each of BA or SA was prepared in water (Milli-Q). The mixture was shaken well until a homogenous and clear solution formed. If necessary, a small amount of methanol was added to help dissolution. The same method was applied to prepare a 500 mg/L internal standard CA stock solution. All stock solutions were covered with aluminum foil, stored in a freezer (4 °C) and away from light (ready for a maximum usage of one month). Before each experiment, BA and SA standard solutions were prepared in the same volumetric flask, and then diluted to five different concentrations in the range 20– 100 mg/L at constant intervals, where 50 mg/L internal standard (CA) was added to every diluted standard solution.
2.3. Sample preparation 2.3.1. Extraction procedure Approximately 1 g of sample was accurately weighed in a 10 mL capped test tube. A 0.5 mL aliquot of the internal standard CA stock solution, 0.5 mL hydrochloric acid–water (1:1, v/v) and 5 mL diethyl ether were then added successively. The mixture was then sonicated for 1 min before being allowed to separate into layers. The upper ether layer was quantitatively transferred by an injector to another 10 mL capped test tube.
2.3.2. Back extraction procedure A 1 mL aliquot of aqueous sodium hydroxide (0.1 M) was added to the diethyl ether extracts. The resulting mixture was sonicated for 1 min, and then allowed to separate into layers. The upper ether layer was discarded, and the lower aqueous layer was placed in a 40 °C water bath to evaporate any residual diethyl ether. Finally, the residue was transferred to a 5 mL volumetric flask and diluted with water to scale.
27
3.2. Optimization of analysis conditions In order to obtain maximum sensitivity, it is optimal to detect each compound at its maximum absorbance wavelength. The UV–vis spectra of the compounds are shown in Fig. 1. It can be anticipated that a small peak would be obtained for BA if the detection wavelength was fixed at 275 nm. On the other hand, small peaks for BA and CA would be obtained if the detection wavelength was set at 235 nm. The maximum peak for BA could be obtained if the detector was set at 225 nm where peak intensities for BA, SA and CA were the same. Therefore, 225 nm was selected as a suitable detection wavelength. In order to verify this prediction, a standard mixture of BA, SA and CA was injected into the HPLC column at maximum absorption wavelengths of 235 and 225 nm separately. As shown in Fig. 2(a), it is evident that the peaks of BA and CA were too small when the detector was set at 235 nm. However, in Fig. 2(b), all peaks were of satisfactory intensity when the detector was set at 225 nm. To protect the environment and the health of the operators, the use of organic solvents (especially highly toxic solvents such as methanol), should be reduced as much as possible. It is generally acknowledged that to minimize matrix effects, the solvent for preparation of samples and standard solutions should be identical. In this work, the extraction-stripping two-step procedure in the sample preparation step eventually drove the sample into water matrix. This allows water to be used for preparing the BA and SA standard solutions, thus decreasing the quantity of organic solvent. To select a suitable internal standard, an extensive literature survey was done. However, no suitable internal standard has been reported so far, for the determination of BA and SA content by HPLC. Phthalate, hendecanoic acid and decylic acid are common internal standards for the determination of BA and SA content in soy sauce by gas chromatography (in China). However, these internal standards were not suitable for the technique in this study. Phthalate could get hydrolyzed by aqueous sodium hydroxide in the back extraction process. Furthermore, hendecanoic acid and decylic acid have poor UV absorption in the 190–400 nm range and are unsuitable for HPLC with an UV detector. Cinnamic acid was selected for this study due to its similar structure with benzoic acid. The initial results were promising, so cinnamic acid was chosen as the internal standard in this new developed method.
2.4. Chromatographic conditions Analytical separation was carried out on an Agilent 1100 HPLC unit using a Supelco 516 C18 column (25 cm 4.6 mm, 5 lm) at room temperature. The detector used was an UV–vis spectrophotometer set at 225 nm and the volume of sample injected was 20 lL. The mobile phase used was methanol–ammonium acetate buffer (0.02 M) (30:70, v/v); the flow rate was 1 mL/min.
3. Results and discussion 3.1. Calibration curve and detection limit The calibration curve was constructed by plotting the peak area ratio of BA or SA and the internal standard CA against the concentration of BA or SA. Excellent linearity was obtained within the concentration range of 20–100 mg/L (the wavelength used to perform the calibration was 225 nm), giving both samples a correlation coefficient of 0.9996. The detection limit of BA, SA and CA was found to be 0.2, 0.1 and 0.5 mg/L respectively in the final solution when the signal-to-noise ratio was greater than 3:1.
Fig. 1. UV spectra of BA, SA and CA (10 mg/L each); the solvent was methanol– ammonium acetate buffer (0.02 M) (30:70, v/v).
28
M. Ding et al. / Food Chemistry 183 (2015) 26–29
Fig. 2. Typical chromatogram of a standard mixture of BA, SA and CA. mg/L: BA, 40; SA, 40; CA, 50. The detector wavelength was set at: (a) 235 nm; (b) 225 nm.
Table 2 BA and SA contents in commercial soy sauce analyzed by HPLC. Analyte Soy Soy Soy Soy b
sauce sauce sauce sauce
1 2 3 4
BA (lg/g) ± SD
SA (lg/g) ± SD
238.7 ± 2.9 336.1 ± 2.7 661.3 ± 5.7 292.8 ± 2.0
246.4 ± 3.1 NDb ND 311.7 ± 2.9
ND, not detectable; SD, standard deviation; n = 3.
away from light). It was found that the relative standard deviation (RSD) values obtained were less than 3%. There was no significant influence of the stability of the preservatives on the experimental results during sample preparation and HPLC analysis. The accuracy of the current method was validated through recovery studies of the preservatives. BA and SA with two concentration levels were added to real samples of different soy sauces, and the resulting mixtures were analyzed by the method developed. The technique provided recovery percentage values from 96.1% to 104.3%, as shown in Table 1.
Fig. 3. Chromatogram of the soy sauce extraction.
3.3. Chromatogram of soy sauce extraction The chromatogram of soy sauce extraction is shown in Fig. 3. As shown in Fig. 3, separation of all the components was achieved in less than 8.1 min. The retention times of BA, SA and CA were 3.654, 4.497 and 8.007 min, respectively.
3.5. Analysis of commercial soy sauce Commercial soy sauce samples were analyzed by the developed procedure. As shown in Table 2, none of the positive samples violate the legal limit of 1.0 g/kg for BA or SA, either independently or in combination.
3.4. Precision and recovery studies 3.6. Compared with the standard method The repeatability of the method was judged by triplicate HPLC analysis with the same soy sauce under the same sample preparation procedure. The reproducibility over different days was tested for the determination of the same standard mixtures, which were stored in a refrigerator and covered by aluminum foil (to keep
To investigate the accuracy of the developed method, the National Standards of the People’s Republic of China (GB/T 23495, 2009) was employed to determine the BA and SA contents in soy sauce (Table 3). As shown in Table 3, the quantitative results
Table 1 Recovery of BA and SA from spiked soy sauce samples analyzed by HPLC.
a
Analyte
Previous (lg/g) ± SDa BA
SA
BA
SA
BA
SA
BA
SA
Soy sauce 1
238.7 ± 2.9
246.4 ± 3.1
Soy sauce 2
336.1 ± 2.7
0
100 200 100 200
100 200 100 200
337.3 ± 2.9 430.9 ± 3.6 434.2 ± 4.9 531.7 ± 4.1
346.7 ± 2.1 444.2 ± 3.8 104.3 ± 2.5 202.6 ± 3.4
98.6 96.1 98.1 97.8
100.3 98.9 104.3 101.3
SD, standard deviation; n = 3.
Added (lg)
Found (lg /g) ± SD
Recovery (%)
M. Ding et al. / Food Chemistry 183 (2015) 26–29 Table 3 Comparison of BA and SA contents found in commercial soy sauce. Analyte
The method developed
Standard method
BA (lg/g) ± SD SA (lg/g) ± SD BA (lg/g) ± SD SA (lg/g) ± SD Soy sauce 1 238.7 ± 2.9 Soy sauce 2 336.1 ± 2.7 Soy sauce 3 661.3 ± 5.7
246.4 ± 3.1 ND ND
241.8 ± 2.2 339.1 ± 3.4 663.3 ± 4.9
246.5 ± 2.6 ND ND
29
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 03.025.
References
SD, standard deviation; n = 3; ND, not detectable.
of three different soy sauce samples are in good agreement with those measured by the standard method. 4. Conclusions A rapid, accurate and environment-friendly procedure was developed for the quantitative determination of BA and SA in soy sauce. Under the recommended conditions, separation of all the components was achieved in less than 8.1 min. Sample preparation includes a two-step procedure: extraction and back extraction. The total amount of organic solvent used was only 5 mL of diethyl ether in sample preparation, and no organic solvent was used in the preparation of the standard solutions. Meanwhile, benzoic and sorbic acids were determined by an internal standard method, which greatly reduced the effects of target losses in extraction and back extraction. Thus the experimental results were quite accurate with the relative standard deviations (RSD) of less than 3% and the recoveries were 96.1–104.3%. These advantages make this method a potential and valuable tool to determine BA and SA quantitatively in soy sauce, which could be used for industrial purposes. Acknowledgement This project was financially supported by Natural Science Foundation of Jiangsu Province of China (BK 20131343 and BK 20141391).
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