Anal. Chem. 2010, 82, 5897–5899
Colorimetric Determination of Melamine in Dairy Products by Fe3O4 Magnetic Nanoparticles-H2O2ABTS Detection System Ning Ding, Na Yan, Cuiling Ren, and Xingguo Chen* National Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, China In this paper, a simple and rapid colorimetric method, which does not require any expensive and complex instruments, is established for the determination of melamine in dairy products. Lower than 2.5 ppm (the safety limit in the USA and EU) of melamine in real samples can be detected exactly with the recoveries in a range from 98-115% using a 721-A spectrophotometer. More significantly, the existence of melamine can be visually evaluated easily without the aid of any instrumentation. Melamine, with a chemical formula of C3H6N6, is used primarily in the synthesis of melamine formaldehyde resins for manufacturing laminates, plastics, coatings, commercial filters, glues or adhesives, dishware, and kitchenware. Because of its high nitrogen level (66% by mass), melamine has been illegally added to dairy products to make a high reading of total nitrogen content as the false measurement of protein level.1,2 Unfortunately, melamine can result in the formation of insoluble crystals in kidneys, thus causing the formation of kidney stones.3,4 Ingestion of melamine at levels above the safety limit (2.5 ppm in the USA and EU; 1.0 ppm for infant formula milk powder in China) may cause kidney failure and even death, particularly for vulnerable individuals such as infants and young children.3-6 Gas chromatography, liquid chromatography, capillary electrophoresis, and low-temperature plasma probe combined with tandem mass spectrometry have been utilized for the determination of melamine.7-18 However, all of these techniques require * Corresponding author. Phone: +86-931-891-2763. Fax: +86-931-891-2582. E-mail:
[email protected]. (1) Wu, Y. N.; Zhao, Y. F.; Li, J. G. Biomed. Environ. Sci. 2009, 22, 95–99, Melamine Analysis Group. (2) Kim, C.-W.; Yun, J.-W.; Bae, I.-H.; Lee, J.-S.; Kang, H.-J.; Joo, K.-M.; Jeong, H.-J.; Chung, J.-H.; Park, Y.-H.; Lim, K.-M. Chem. Res. Toxicol. 2010, 23, 220–227. (3) Langman, C. B.; Alon, U.; Ingelfinger, J.; Englund, M.; Saland, J.; Somers, M. J. G.; Stapleton, F. B.; Sibu´, N. O.; Cochat, P.; Wong, W.; Eke, F. U.; Satlin, L.; Salusky, I. Pediatr Nephrol. 2009, 24, 1263–1266. (4) Ching, W. L.; Lawrence, L.; Xiao, Y. C.; Sidney, T.; Samson, S. Y. W.; et al. Clin. Chim. Acta 2009, 402, 150–155. (5) Ai, K. L.; Liu, Y. L.; Lu, L. H. J. Am. Chem. Soc. 2009, 27, 9496–9497. (6) Brown, C. A.; Jeong, K. S.; Poppenga, R. H. J. Vet. Diagn. Invest. 2007, 19, 525–531. (7) Yokley, R. A.; Mayer, L. C.; Rezaaiyan, R.; Manuli, M. E.; Cheung, M. W. J. Agric. Food Chem. 2000, 48, 3352–3358. (8) Yan, N.; Zhou, L.; Zhu, Z. F.; Chen, X. G. J. Agric. Food Chem. 2009, 57, 807–811. 10.1021/ac100597s 2010 American Chemical Society Published on Web 06/07/2010
expensive, complicated instruments and make on-site, real-time melamine testing difficult.5 Accordingly, there is an urgent need to establish a simple and highly sensitive assay method for the detection of melamine in dairy products without dependence on expensive and complex instruments. As UV-vis spectrophotometer is the most common analytical instrument, a colorimetric method which can detect melamine quickly and sensitively is very necessary for the safety testing of food. A recent research reported that Fe3O4 magnetic nanoparticles (MNPs) exhibited an intrinsic enzyme mimetic activity which could be used to detect hydrogen peroxide.19 According to this character, Wei and Wang20 studied the catalytic effect of Fe3O4 MNPs on the reaction between 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) and H2O2, and established a colorimetric method for the determination of H2O2. The detecting limit of H2O2 was as low as 3 × 10-6 M, which was quite sensitive. Mixing of melamine and hydrogen peroxide generates an addition compound21-23 (see Figure 1), which is stable under 100 °C according to the thermal analysis results reported by Nagaishi (9) Ding, T.; Xu, J.; Li, J.; Shen, C.; Wu, B.; et al. Chin. J. Chromatogr. 2008, 26, 6–9. (10) Cai, Q.; Ouyang, Y.; Qian, Z.; Peng, Y. C. J. Chromatogr. 2008, 26, 339– 342. (11) Filigenzi, M. S.; Puschner, B.; Aston, L. S.; Poppenga, R. H. J. Agric. Food Chem. 2008, 56, 7593–7599. (12) Andersen, W. C.; Turnipseed, S. B.; Karbiwnyk, C. M.; Clark, S. B.; Madson, M. R.; Gieseker, C. M.; Miller, R. A.; Rummel, N. G.; Reimschuessel, R. J. Agric. Food Chem. 2008, 56, 4340–4347. (13) Filigenzi, S. M.; Tor, R. E.; Poppenga, H. R.; Aston, A. L.; Puschner, B. Mass Spectrom. 2007, 21, 4027–4032. (14) Karbiwnyk, C. M.; Andersen, W. C.; Turnipseed, S. B.; Storey, J. M.; Madson, M. R.; et al. Anal. Chim. Acta 2008, 637, 101–111. (15) Baynes, R. E.; Smith, G.; Mason, S. E.; Barrett, E.; Barlow, B. M.; et al. Food Chem. Toxicol. 2008, 46, 1196–1200. ´ . J.; Herna´dez, F. Anal. Chim. (16) Sancho, J. V.; Iba´n ˜ez, M.; Grimalt, S.; Pozo, O Acta 2005, 530, 237–243. (17) Patakioutas, G.; Savvas, D.; Matakoulis, C.; Sakellarides, T.; Albanis, T. J. Agric. Food Chem. 2007, 55, 9928–9935. (18) Scheepers, M. L.; Meier, R. J.; Markwort, L.; Gelan, J. M.; Vanderzande, D. J.; et al. J. Vib. Spectrosc. 1995, 9, 139–146. (19) Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; et al. Nat. Nanotechnol. 2007, 2, 577–583. (20) Wei, H.; Wang, E. K. Anal. Chem. 2008, 80, 2250–2254. (21) Wielicka, J.; Ptasiewicz-Malinowska, A.; Jedrych, T.; Minda, D.; Wiejak, B.; et al. Pol. J. Chem. Technol 2003, 5, 19–21. (22) Chehardoli, G.; Zolfigol, M. A. Phosphorus Sulfur 2010, 185, 193–203. (23) Dirscherl, W.; Moersler, B. Ann. Chem. 1964, 677, 177–184.
Analytical Chemistry, Vol. 82, No. 13, July 1, 2010
5897
Figure 1. The reaction of melamine and H2O2.
et al.24 On the basis of this reaction and the system described above, we established a new method for the determination of trace amount of melamine in dairy products by measuring the wastage of H2O2. In this method, the existence of melamine can cause a color change of the reaction system, and the color change can be visually observed directly. The results indicate that this method is simple and rapid in the detection of melamine without requiring any expensive and complicated instruments. EXPERIMENTAL SECTION Chemicals and Materials. Ferric chloride and ferrous chloride was purchased from Beijing Chemical Reagent Company (Beijing, China). H2O2 (30 wt %), hydrochloric acid (36-38 wt %), and ammonium hydroxide (25-28 wt %) were purchased from Baiyin Liang You Chemicals Reagent Co. LTD (Baiyin, China). 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich. Melamine was purchased from Shanghai Shi Yi Chemicals Reagent Co. LTD (Shanghai, China). Other reagents were all analytical reagent grade. Distilled water was used throughout the experiments. Instrument. A 721-A spectrophotometer (Sichuan Analysis Apparatus Factory, China) was used to record absorption spectra and measure absorbance. Preparation of Fe3O4 MNPs. Fe3O4 MNPs were prepared according to the reported method.20 First, 5 mL of 1 M ferric chloride aqueous solution in 2 M HCl and 1 mL of 2 M ferrous chloride aqueous solution in 2 M HCl were prepared. Second, the two solutions were mixed and deoxygenated by purging with nitrogen gas for 10 min. Third, 50 mL of 0.7 M ammonia solution was added into the mixed solution, and the final solution was stirred for 30 min at room temperature in anitrogen atmosphere. The formed Fe3O4 colloidal particles were washed three times with water. Finally, the Fe3O4 MNPs were kept in a nitrogen atmosphere until dry, and 0.16 g of dry Fe3O4 MNPs powder was redispersed in water for use. Sample Preparation. The raw milk and milk powder were purchased from local supermarkets. First, 1 mL of acetonitrile, 1 mL of CCl3COOH, and 7 mL (for raw milk)/9 mL (for milk powder) of water were added into 2.0 mL of raw milk or 1.0 g of milk powder. Then the mixed liquor in a cuvette was ultrasonically extracted for 15 min and then centrifugated at 2500 rpm for 10 min. The obtained supernatant was used for the following detection. Detection of Melamine. The samples were determined according to the following steps: (a) 24.0 µL of the samples solution, 24.0 µL of 0.1 M H2O2, 24.0 µL of 0.06 M ABTS, and 10.0 µL of Fe3O4 MNPs stock solution were added into 185.0 µL of 0.2 M acetic acid-acetate buffer (pH 4.0). (b) The mixed (24) Nagaishi, T.; Matsumoto, M.; Yoshigana, S. J. Therm. Anal. 1990, 36, 55– 60.
5898
Analytical Chemistry, Vol. 82, No. 13, July 1, 2010
Figure 2. Typical photograph of different concentrations of melamine standard solutions under the optimum conditions (from left to right: the concentration of melamine in water is B, 0 M; S1, 8.0 × 10-7 M; S2, 2.0 × 10-6 M; S3, 4.0 × 10-6 M; S4, 8.0 × 10-6 M; S5, 2.0 × 10-5 M; S6, 4.0 × 10-5 M.
solution was incubated in a 45 °C water bath for 10 min to hold the reaction and then kept in an ice-water bath for 10 min to stop the reaction completely. (c) The Fe3O4 MNPs were then removed from the reaction solution by an external magnetic field; (d) 200 µL of the resulting solution was added into 2.8 mL of water in a 10 mL cuvette and used for absorbance measurement at wavelength 417 nm.20 Calibration Curves. Different amounts of melamine were added to 2.0 mL of melamine-free raw milk and also to 1.0 g of milk powder corresponding to a final concentration of melamine in them as 0.8, 2.0, 4.0, 8.0, 20.0, and 40.0 µM. The raw milk and milk powder spiked melamine samples were handled as the steps described in the Sample Preparation section. Then the obtained solutions were tested for fitting the regression equation of melamine. RESULTS AND DISCUSSION Mechanism of the Sensing System. Two reactions were involved for the determination of melamine in the dairy products. For the reaction of melamine and H2O2, the influence of interference can be eliminated by fitting a regression equation of melamine based on the matrix of raw milk and milk powder, respectively. In addition, the reaction of H2O2 and ABTS using Fe3O4 MNPs as catalyst was selective.20 Therefore, melamine can be detected selectively by measuring the wastage of H2O2. Optimization of Experimental Conditions. The conditions including the concentration of ABTS (30-70 mM), the concentration of H2O2 (0.001-0.05 M), the pH of the reaction buffer (3.0-8.0), the incubation temperature (25-65 °C), and the reaction time (1-20 min) were examined in detail. The results showed that the optimum conditions were as follows: the concentration of ABTS was 60 mM, the concentration of H2O2 was 0.01 M, the pH of the reaction buffer was 4.0, the incubation temperature was 45 °C, and the reaction time was 10 min. The other experimental conditions were the same as the previous report20 to ensure the accurate detection of the wastage of H2O2. A typical photograph of different concentrations of melamine standard solutions under the optimum conditions is shown in Figure 2. The gradual color change can be visually identified easily. Method Validation and Application. Calibration Curves and Performance Characteristics. To eliminate the interference of matrix, the calibration curves were prepared based on the raw milk matrix and the powder milk matrix. The results are sum-
Table 1. Information on the Concentration-Response Curvesa matrix
linear range (µM)
regression equation
correlation coefficient
raw milk milk powder
2.0-40.0 2.0-40.0
A ) -0.0083ca - 0.136 A ) -0.0085ca - 0.152
0.9903 0.9903
a
A, absorbance; c, the concentration of melamine (µM).
Table 2. Results of the Determination of the Melamine in Raw Milk raw milk
original amount (µM)
added (µM)
found (µM)
recovery (%)
m1 m2
0 0
8.0 20.0
8.9 19.7
111 98
Table 3. Results of the Determination of the Melamine in Milk Powder milk powder
original amount (µM)
added (µM)
found (µM)
recovery (%)
p1 p2
0 0
8.0 20.0
9.2 19.7
115 98
marized in Table 1. The blank matrix was free of melamine, which has been proved by capillary zone electrophoresis8 (data not shown). The results indicated that the lowest concentration of detectible melamine in raw milk and milk powder could be 0.25 ppm (2.0 µM), which was much lower than the U.S. Food and Drug Administration estimated melamine safety limit of 2.5 ppm (20 µM) and the safety limit of 1.0 ppm (8.0 µM) in China.5 Analytical Applications. Under the optimized conditions, the amounts of melamine in raw milk and milk powder were determined. The results are shown in Tables 2 and 3. The recoveries of the 1.0 ppm/8 µM of melamine (safety limit in China) were 111% with raw milk and 115% with milk powder, respectively. Figures 3 and 4 show the good colorimetric differentiation. CONCLUSION In summary, the colorimetric determination of melamine in dairy products by the Fe3O4 MNPs-H2O2-ABTS detection system was accurate, reliable, sensitive, and convenient. Using the Fe3O4 MNPs-H2O2-ABTS system, the dairy products contained melamine above the safety limits could also be easily detected visually. The whole process was achieved in less than 1 h from the beginning of sampling to the final step of obtaining the results from the spectrophotometer. The convenient and
Figure 3. Typical photograph for melamine detection in the raw milk (from left to right: the concentration of melamine in the raw milk is mB (milk blank), 0 M; m1 (milk sample 1), 8.0 × 10-6 M/1.0 ppm; m2 (milk sample 2), 2.0 × 10-5 M/2.5 ppm.)
Figure 4. Typical photograph for melamine detection in the milk powder (from left to right: the concentration of melamine in the milk powder is pB (milk powder blank), 0 M; p1 (milk powder 1), 8.0 × 10-6 M/1.0 ppm; p2 (milk powder 2), 2.0 × 10-5 M/2.5 ppm.
reliable color examination for the melamine content in the milk sample enables a practical application of the proposed method to real dairy products. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 20875040). Received for review March 5, 2010. Accepted May 29, 2010. AC100597S
Analytical Chemistry, Vol. 82, No. 13, July 1, 2010
5899
