Available online: 27 May 2011

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1 This article was downloaded by: [T&F Internal Users], [Cathy Coleman] On: 30 June 2011, At: 03:24 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: Analysis of melamine migration from melamine food contact articles Z. Chik a, D.E. Mohamad Haron a, E.D. Ahmad a, H. Taha a & A.M. Mustafa a a Shimadzu-UMMC Centre for Xenobiotics Studies (SUCXeS), Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Available online: 27 May 2011 To cite this article: Z. Chik, D.E. Mohamad Haron, E.D. Ahmad, H. Taha & A.M. Mustafa (2011): Analysis of melamine migration from melamine food contact articles, Food Additives & Contaminants: Part A, 28:7, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan, sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Food Additives and Contaminants Vol. 28, No. 7, July 2011, Analysis of melamine migration from melamine food contact articles Z. Chik*, D.E. Mohamad Haron, E.D. Ahmad, H. Taha and A.M. Mustafa Shimadzu-UMMC Centre for Xenobiotics Studies (SUCXeS), Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia (Received 19 September 2010; final version received 22 March 2011) Migration of melamine has been determined for 41 types of retail melamine-ware products in Malaysia. This study was initiated by the Ministry of Health, Malaysia, in the midst of public anxiety on the possibility of melamine leaching into foods that come into contact with the melamine-ware. Thus, the objective of this study was to investigate the level of melamine migration in melamine utensils available on the market. Samples of melamine tableware, including cups and plates, forks and spoons, tumblers, bowls, etc., were collected from various retail outlets. Following the test guidelines for melamine migration set by the European Committee for Standardisation (CEN 2004) with some modifications, the samples were exposed to two types of food simulants (3% acetic acid and distilled water) at three test conditions (25 C (room temperature), 70 and 100 C) for 30 min. Melamine analysis was carried out using LC MS/MS with a HILIC column and mobile phase consisting of ammonium acetate/formic acid (0.05%) in water and ammonium acetate/formic acid (0.05%) in acetonitrile (95 : 5, v/v). The limit of quantification (LOQ) was 5 ng/ml. Melamine migration was detected from all samples. For the articles tested with distilled water, melamine migration were [median (interquartile range)] 22.2 (32.6), 49.3 (50.9), 84.9 (89.9) ng/ml at room temperature (25 C), 70 and 100 C, respectively. In 3% acetic acid, melamine migration was 31.5 (35.7), 81.5 (76.2), (126.7) ng/ml at room temperature (25 C), 70 and 100 C, respectively. This study suggests that excessive heat and acidity may directly affect melamine migration from melamine-ware products. However the results showed that melamine migration in the tested items were well below the specific migration limit (SML) of 30 mg/kg (30,000 ng/ml) set out in European Commission Directive 2002/72/EC. Keywords: LC/MS; food contact materials; food simulants; organic foods Introduction Melamine is a nitrogen-rich heterocyclic triazine with the chemical formula C 3 H 6 N 6 and chemical structure 1,3,5-triazine-2,4,6-triamine (Figure 1). Melamine has many uses in industrial applications, such as in fire retardant products, adhesives for woods, molding compounds and fertilizer urea mixtures (Johannes 2005). Melamine resin or melamine formaldehyde is widely used in the manufacturing of plastic-ware because it is heat-resistant and durable (Lokensgard and Richardson 2003). However, melamine-ware, though non-flammable, can decompose under extreme heat; thus, at temperature above 300 C, it is not suitable for use in the conventional ovens (Jacaab 2007). Over the years, many issues have been raised on the migration of chemical substances into foods during packaging, and chemical migration from food contact materials and utensil (WHO 2009). This is a growing concern because foods and beverages can act strongly with the materials in contact under multiple conditions or environments. Ingelfinger (2008) demonstrated that melamine, in particular, can migrate into food in a measureable amount when leached by acid. Currently, there is an increasing public awareness of melamine contamination in foods since the 2007 incident where melamine-tainted pet food caused renal failure and death in cats and dogs in the United States and Europe (Brown et al. 2007). Another major incident in China in 2008, causing death and inflicting serious kidney damage in infants and children who consumed melamine-tainted milk formulae, has increased fear and anxiety among the public (Gossner 2009). Melamine toxicity has been reported in animal studies, such as bladder stones and urinary bladder N H 2 N NH 2 N NH 2 Figure 1. Structural formula of melamine. N *Corresponding author. zamrichik@um.edu.my ISSN print/issn online ß 2011 Taylor & Francis DOI: /

3 968 Z. Chik et al. tumors in animal tests after long exposure to melamine (IARC 1999, Bingham et al. 2001). However, as yet, there are no direct studies involving human subjects on the adverse effect of melamine on the renal system. The migration limit for melamine was established by the European Union Plastics Directive at 30 mg/kg (Ishiwata et al. 1986). Lund et al. (2006) and Takiko et al. (1990) have shown that factors such as temperature, acidity and repeated use of melamine plastics greatly affected the level of melamine migration. A survey on melamine articles in UK to determine the specific migration limit (SML) in melamine-ware articles found that the migration level was well below the limit (30 mg/kg) set by the European Union Plastics Directive (Bradley et al. 2005). Another recent study in China found that three of six melamine resin containers showed positive melamine migration in a smaller amount (Lu et al. 2009). In general, melamine-ware is safe to use within the acceptable temperature range ( 30 to þ120 C, and up to 140 C) according to the manufacturer s instructions, as long as it is not used to store acidic foods or overheated, especially when used in conventional or microwave ovens. There may be some cases where the melamine wares are wrongly used and mishandled, or not manufactured according to the certified standard specifications. In Malaysia, melamine-ware, such as plates, ladles, spoons, trays, cups and bowls, are widely used. These items are commonly used to serve food at home and at food stalls because they are attractive, relatively cheap, lightweight and durable. However, after the recent melamine in milk scare, there were mixed reactions among Malaysian consumers about the safety of using melamine wares in everyday life. Therefore, in the interest of public safety, this study was carried out to evaluate the level of melamine migration in melamineware products marketed in Malaysia. Materials and methods Chemical and reagents HPLC-grade acetonitrile was purchased from Fisher (USA), formic acid (85%) was purchased from BDH (Poole, Dorset, UK) and melamine standard (99.0% purity) was purchased from Sigma-Aldrich (Malaysia). All chemicals used were of analytical grade. Test items A total of 246 samples of 41 types of melamine-ware products, comprising bowls, spoons, plates, cups and saucers, tumblers, trays and chopsticks, were collected. For each type of test article, one sample was tested for each of the test condition, amounting to six samples per product. Instrumentation and chromatographic system The LC system (Shimadzu LC-20AD) consisted of a binary pump and autosampler (SIL 20AC 200). Sample injection was performed by using a Shimadzu SIL20AC 200 auto injector. Mass spectrometric detection was performed on a Triple Quadrupole mass spectrometer (API SCIEX API3200) with an ion source temperature of 400 C. A Windows XP computer running API SCIEX Analyst Software (Version 1.4.2) was used for data acquisition and processing. Chromatographic separation was performed at 40 C (Shimadzu CTO-20AC) using an Altima HP HILIC C 18 column (3 m, 150 mm long 2.1 mm) protected by a guard filter (5 um). The mobile phase consisted of pump (A) 10 mm ammonium acetate with 0.05% formic acid in water and 10 mm ammonium acetate with 0.05% formic acid in acetonitrile (95 : 5, v/v). Gradient elution was used to elute the compounds at a flow rate of 0.25 ml/min with a total run time of 6 min. The gradient programme began at 0.1 min for pump B (80%), then decreases at 5%, hold for 2 min, and finally returning to 80% at 2.01 min and hold until 6 min. Analytes were detected in the multiple reaction monitoring mode (MRM) using electrospray positive ionization (ESI positive). The monitored precursor ion for melamine was m/z , the most sensitive ion in the Q1 mass spectra. However, after fragmentation, the MS/MS daughter ion spectra indicated that the more intense product fragment was m/z for melamine. The dwell time was 0.1 s. Nitrogen was used as the collision gas. The NM20ZA high purity nitrogen and air generators were supplied by Peak Scientific Instruments. Assay procedure and method Preparation of stock and standard solutions Stock solutions were prepared in acetonitrile/water mixture (1 : 1, v/v) at a concentration of 1 mg/ml. The working solution was diluted from the stock solution to obtain a concentration of 50 mg/ml. All calibrators were prepared by serial dilution of the working solution. The standard solutions were stored in the dark at 4 C before analysis. Extraction procedure The 246 samples of 41 type of melamine products were exposed to distilled water and 3% acetic acid at three different test conditions (25, 70 and 100 C) for 30 min. In this study, acetic acid and distilled water were used as food simulants. Samples were extracted by using liquid solid extraction. A 50-ml aliquot of food simulant was pre-heated prior to exposure to samples. Samples were exposed to food simulant by filling

4 Food Additives and Contaminants 969 or immersing the samples with or in the food simulants. Then, 1 ml of food simulant was transferred into a test tube and 10 ml injected into the LC MS/MS. The limit of quantification (LOQ) of this method showing the least concentration of melamine that could be determined was 5 ng/ml. Calibration curve The calibration curve with a range from 5 to 1000 ng/ml was prepared in acetonitrile in three batches. The calibration curve was obtained by plotting area ratios of melamine standard against solvent concentration. All results were calculated using a 1/x weighted quadratic. The peak area, regression coefficient and parameters of the calibration line were calculated from the peak, and data processed by Analyst Software (1.4.2 version; Applied Biosystem, Concord, Canada). The regression coefficient for all the calibration curves were greater than Results The quantification of melamine migration was calculated according to the formula described in a Lu et al. (2009). Melamine migration was detected from Table 1. Melamine levels in test items exposed to two types of food simulants under three different test conditions. Distilled water (ng/ml) 3% Acetic acid (ng/ml) Item Code Sample description 25 C 70 C 100 C 25 C 70 C 100 C 01 MEL 2405 Cereal Bowl MEL 2406 Side Plate 6" MEL 2407 Oval Platter MEL 2408 Deep Soup Plate MEL 2409 Dinner Plate MEL 2410 Rice Bowl MEL 2411 Soup Plate MEL 2412 Bowl MEL 2413 Small Plate MEL 2414 Side Plate 7" MEL 2415 Oval Platter 10" MEL 2416 Rice Scoop MEL 2417 Compartment Tray MEL 2418 Tumbler MEL " Soup Plate MEL 2420 Tableware Spoon MEL 2421 Bowl MEL 2422 Plastic tumbler MEL 2423 Ladle MEL 2424 Chopstick ND ND ND MEL 2425 Fork MEL " Tray MEL 2427 Rim Round Plate MEL 2428 Round Rice Bowl MEL 2429 Tea Cup MEL 2430 Spoon ND MEL 2431 Fork MEL 2432 Rice Scoop MEL 2433 Tray ND MEL 2434 Chopstick MEL 2435 Chili Dish MEL 2436 Tumbler MEL 2437 Plate MEL 2438 Bowl MEL 2439 Cup MEL 2440 Plastic tumbler MEL 2441 Spoon MEL 2442 Fork MEL 2443 Ladle ND MEL " Tray ND MEL 2445 Chopstick Note: ND, not detected.

5 970 Z. Chik et al. all 246 samples of 41 type of melamine ware tested. It was found that the level of melamine migration increased with increasing temperature for both distilled water and acetic acid. For the articles tested with distilled water, melamine migration was (median (IQR)) 22.2 (32.6), 49.3 (50.9), 84.9 (89.9) ng/ml at room temperature (25 C), 70 and 100 C, respectively. In acetic acid, melamine migration were 31.5 (35.7), Figure 2. Melamine levels in test items exposed to distilled water at room temperature (25 C). Figure 3. Melamine levels in test items exposed to distilled water at 100 C.

6 Food Additives and Contaminants 971 Figure 4. Melamine levels in test items exposed to 3% acetic acid at room temperature (25 C). Figure 5. Melamine levels in test items exposed to 3% acetic acid at 100 C.

7 972 Z. Chik et al. Figure 6. Overview of melamine migration in test items relative to the specific migration limit (SML) (76.2), (126.7) ng/ml at room temperature (25 C), 70 and 100 C, respectively. Table 1 summarises the level of melamine detected in all the samples tested. Graphical representations are illustrated in Figures 2, 3, 4 and 5, respectively. for melamine level in test items exposed to 25 C in water, 100 C in water, 25 C in 3% acetic acid and 100 C in acetic acid. Discussion In this study, the variable parameters that cannot be accounted for are the quality and grade of the melamine resin used in the manufacture of the melamine wares. Nevertheless, it can be concluded that the migration level of melamine is directly affected by 3% acetic acid and high temperature. The level of melamine migration showed an increasing pattern from distilled water to acidic conditions and from room temperature to 100 C in most samples. These facts may indicate that melamine-ware users may be exposed to melamine under contact conditions such as acidic conditions and extreme heat. Also, the total surface area of the contact material may suggest a higher amount of melamine migration. In this study, we had difficulties in measuring the area of exposure to the food simulants due to the various shapes of the test items. Therefore, only the amount of food simulant applied was recorded and standardised. Overall results showed that the levels of melamine migration in the tested items were well below the specific migration limit of 30 mg/kg (30,000 ng/ml) set by European Commission Directive 2002/72/EC (CEN 2004) as shown in Figure 6. In view of this, it would be appropriate for health authorities to standardise the international limit and regulations for melamine migration from food contact materials. Our results clearly indicate the possibility of human exposure to melamine through migration from food contact materials. Acknowledgements We would like to thank the Ministry of Health, Malaysia for providing us the samples and the following people, Putri, Zana, Atiqah, and Aziz for their valuable assistance. References Bingham E, Cohrssen B, Powell CH Patty s Toxicology. 5th ed. Vol New York: Wiley. p. 4:1335. Bradley EL, Boughtflower V, Smith TL, Speck DR, Castle L Survey of the migration of melamine and

8 formaldehyde from melamine food contact articles available on the UK market. Food Addit Contam. 22: Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, Kang KI, Sum S, Cistola AM, Brown SA Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in (2004) and (2007). J Vet Diag Invest. 19: CEN EN Materials and articles in contact with foodstuffs. Plastics substances subject to limitation. Part 1: Guide to test methods for the specific migration of substances from plastics to foods and food simulants and the determination of substances in plastics and the selection of conditions and exposure to food simulants. Available from: Gossner CME, Schlundt J, Embarek PB, Hird S, Lo-Fo- Wong D, Beltran JJO, Teoh KN, Tritscher A The melamine incident: Implications for international food and feed safety. Environ Health Perspect. 117: IARC (International Agency for Research on Cancer) Some chemicals that cause tumours of the kidney, or urinary bladder in rodents and some other substances. IARC Monogr Eval Carcinogen Risks Hum. 73: Ingelfinger JR Melamine and the global implications of food contamination. N Engl J Med. 359: Ishiwata H, Inoue T, Tanimura A Migration of melamine and formaldehyde from tableware made of melamine resin. Food Addit Contam. 3: Food Additives and Contaminants 973 Jacaab Jacaab micronized melamine J-40 technical data sheet [Internet]. JACAAB L.L.C. Manufacturing & Repackaging of Specialty Chemicals; [cited 2007 June 1]. Available from: 20Melamine%20J-40%20TDS.pdf Johannes KF Reactive polymers fundamentals and applications: a concise guide to industrial polymers. Norwich (NY): William Andrew Publishing/Plastic Design Library. Lokensgard E, Richardson TL Industrial plastics: theory and application. 3rd ed. Clifton Park (NY): Delmar Learning. Lu J, Xiao J, Yang D-J, Wang Z-T, Jiang D-G, Fang C-R, Yang J Study on migration of melamine from food packaging materials on markets. Biomed Environ Sci. 22: Lund KH, Petersen JH Migration of formaldehyde and melamine monomers from kitchen and tableware made of melamine plastic. Food Addit Contam. 23: Takiko S, Ishiwata H, Yoshihira K Release of formaldehyde and melamine from tableware made of melamine-formaldehyde resin. Food Addit Contam A. 7: WHO Toxicological and health aspects of melamine and cyanuric acid. Report of a WHO Expert Meeting in collaboration with FAO supported by Health Canada. Geneva: World Health Organization.