Project++Melamine+Contamination,+an+Overview

[26] ||
 * **Melamine** ||
 * [[image:http://www.chemspider.com/ImagesHandler.ashx?id=7667]] [26]
 * IUPAC name || 1,3,5-Triazine-2,4,6-triamine ||
 * **Properties** ||
 * Molecular Formula || C3H6N6 ||
 * Molecular Weight || 126.12 g/mol ||
 * Appearance || White Solid [1] ||
 * Density || 1.66 g/cm3 [26] ||
 * Boiling Point || 557.5°C / Sublime [7][26] ||
 * Melting Point || 345°C /Decompose[1][7] ||
 * Flash Point || 325.3°C[26] ||
 * Solubility || 3 g/L (20°C) [27] ||
 * Stability || Stable. Incompatible with strong acids, strong oxidizing agents. Nonflammable [27] ||
 * Toxicity || Harmful if swallowed, inhaled or absorbed through the skin. Chronic exposure may cause cancer or reproductive damage. Eye, skin and respiratory irritant. [7] ||

Introduction
Melamine is an organic compound that contains 66% nitrogen, when it is used in synthesis of melamine formaldehyde resins it has fire retardant properties because of release of the nitrogen gas when burned [1]. The production of melamine is mainly in China, as the consumption and the production of melamine grew considerably between the late 1990s and early 2000s, and the production was reported to be in “serious surplus” by early 2006 [1]. Melamine can be used to manufacture glues or adhesive, plastic, dishware and kitchenware. Melamine is also present from the breakdown of the pesticide cyromazine, and it is approved to be used in many countries [6]. Melamine was once used as a fertilizer for crops because of the rich nitrogen content [2], however because melamine is much more expensive to produce compare to other fertilizer, and the purpose of fertilizer is to quickly release the plant’s nutrient, which the nitrogen mineralization process for melamine is very slow [1].Melamine was also used in a drug to cure African sleeping sickness. Melamine was never used in food otherwise, but since there are plastic cups that contain melamine, sometimes it is consumed. Different countries have different intake limit, and the FDA’s daily intake limit was 0.63 milligram per kilogram body weight per day (mg/kg bw/d) [3]. In 2007 it was discovered that pet food imported from China had melamine adulteration, which caused thousands of pet’s death in the US [4]. It was soon after such incident melamine was also found in milk and different kinds of dairy product, such as liquid milk, powder milk, and even eggs. Since there are other food that is prepared by milk, the melamine contamination affected numbers of other food and drinks, such as cookies, cheesecake, various sweets and snacks and chocolate [5]. Most importantly, since milk is the main food source for infant, the melamine contamination in dairy caused many deaths in infants in China.

Melamine contamination
Since melamine has 66% of nitrogen, the nitrogen content is similar to some protein molecules. The Kjedahl [4] and Dumas [1] tests were used to test the protein content in milk. The Chinese milk producer added water to the milk to increase the volume, and then added melamine to the milk to inflate the protein reading in the adulterated milk to pass the test. Melamine was not tested in dairy products at that time because regulators did not suspect the use of melamine in dairy products yet [4]. Although melamine was considered to be minimally toxic, harmful if swallowed, inhaled or absorbed through skin [7], enough melamine consumed daily became lethal to both animals and human. Out of all the milk producers, Sanlu was the one that had the most amount of melamine added to their dairy product, with a range from <0.05 to 4700mg/kg melamine in infant formula powder milk [8], and other dairy products have level of melamine ranged from 0.09 to 6196.61 mg/kg[6]. According to the suggested daily intake on the Sanlu infant formula and the amount of melamine that was present in the powdered milk, infants that is aged from 3-24 months could consume up to 22.3 to 61.0 mg/kg bw/d [8], which is much higher compared to the FDA suggested daily intake, which was 0.63mg/kg bw/d and it is suggested for adult. Aside from melamine, the analogs of melamine such as cyanuric acid, ammeline and ammelide were also found in these dairy products [6]. There are numbers of other ways for human to be exposed to melamine and its analogues, such as the pesticide cyromazine, or the plastic food container and cups. In some countries melamine was used illegally as an animal feed for chickens, pigs and fishes, and there might be a carryover effect for human although there are no quantitative data that exist [6]. In this regard, the melamine contamination could probably affect the entire food chain.

Toxicity of Melamine
Since melamine is not a natural product it is not approved to for its addition in food. Chronic exposure of melamine may cause cancer or reproductive damage [7]. When the toxicity of melamine and cyanuric acid when consumed orally were tested on mice, the common effects were weight loss, bladder and kidney stones, crysatlluria, and epithelial hyperplasia of urinary bladder [4]. Melamine was also tested for its carcinogenicity, and it showed that prolonged period of exposure of melamine caused bladder tumor and urinary bladder hyperplasia in male mice, and continuous dietary exposure of melamine will increase the production of bladder stones and the occurrence of urinary bladder tumor [4]. When the experiment was performed on cat, it was shown that melamine or cyanuric acid alone does not have any effects on the renal function, but the combination of the two caused acute renal failure 48 hours after the consumption [4]. A research suggested that melamine cyanurate could be more toxic than either melamine or cyanuric acid alone [1]. Melamine does not build up in animal body as it is excreted out about 10-15 days after ingestion. The reason for the cause of the bladder stone is hypothesized that since melamine has a poor solubility in water, the melamine cyanurate were absorbed through the gastrointestinal tract and distributed to the kidneys to form kidney stones; however this has not been confirmed. During the melamine contamination incidence, there were numbers of infant that was affected by the melamine milk. A study (Ching-wan Lam et al. [9]) was performed in Hong Kong and reviewed by the University of Hong Kong and Hospital Authority [9]. Patients with signs of urinary tract stones after the consumption of melamine contaminated milk has shown a list of symptoms including abnormal renal functions, bilateral hydronephrosis, hydronephrosis in the kidneys and acute kidney failure [9]. In the same study it was shown that unlike animals, only 1 out of 15 patients that participated in the study had melamine crystals in the urine [4][9], and it is found that the amount of melamine consumed correlate to the size of the kidney stones. Unlike the animals, there was no correlation between levels of cyanuric acid and the size of the kidney stones, which implied that cyanuric acid could be not important for the formation of the stones. The kidney stones caused by melamine consumption are usually small, soft and multiple. Non-invasive treatment and hydration is usually effective for treatment, and even if surgery is necessary it is usually successful [9]. Children that were affected by the melamine contaminated milk were usually younger, so it is possible that infant and young children are at a higher risk because of their immature metabolic and excretory mechanisms [9]. A study (Jinyu Ma et al. [12]) has shown that melamine can interfere with the Maillard reaction. The maillard reaction is a chemical reaction between amino acid and a reducing sugar that requires heat, which is responsible for browning of the food [12][13]. Melamine can directly react with the reducing sugar and lower the contents of aldehyde in the Maillard models. It also showed that this interference can give rise the new chemical entities which could potentially be toxic [12], however the real effects of this involvement of melamine in the Maillard reaction still need to be further studied.

Risk assessment
The AQSIQ collected and inspected all types of milk and dairy product, with the limit of quantitation being 0.05 mg/kg and any sample above that level is considered to be positive. Before September 14, 2008, 22 out of 109 producers of infant formula powdered milk was found positive with the maximum of 2563 mg/kg melamine per milk; 20 out of 154 producer of regular powered milk was found positive with the maximum of 6196 mg/kg melamine per milk and 3 out of 5 producers of liquid milk were found positive with a maximum of 8.6 mg/kg [6]. However after September 14, 2008 testing for all of the powdered milk products was negative. The AQSIQ has set the new interim control limit in food product (2.5mg/kg) and infant formula (1 mg/kg). In Hong Kong the safe limit for infant under the age of 3 months and lactating women are 1 mg/kg [1][6]. As for Taiwan, the Department of Health in Taiwan decided to follow actions taken by the WHO and Hong Kong, due to the fact that it is difficult to quickly examine food samples for less than 2ppm of melamine, the standard of minimum TDI is set to 2.5ppm. Currently any dairy products with melamine at all are banned from Taiwan [11].
 * The General Administration of Quality Supervision, Inspection and Quarantine of China (AQSIQ)**

Before the melamine contamination incident the suggested maximum daily intake was 0.63 mg/kg bw/d. This number was determined an experiment that consist of 10 young male rats and a 13-week toxicity assay, with diet of different levels of melamine. The dose level of 63 mg/kg bw/d is the no-observed-adverse-effect level(NOAEL) [3]. This number is then divided by a safety factor of 100, which then becomes the TDI of 0.63 mg/kg bw/d. After the melamine contamination, the FDA has increased the safety factor by tenfold, which becomes the current TDI, 0.0631 mg/kg bw/d [3][4]. The US FDA stated that they will examine all proteins containing product for melamine contamination, and if the product is contaminated with melamine or its analog, the FDA will take action to prevent the products from entering the U.S. food supply [10].
 * US food and drug administration (FDA)**

The WHO performed a similar analysis much like the FDA, with data obtained from the 13 weeks study and determined that the NOAEL was 35 mg/kg bw/d. The WHO used a safety factor of 200 which gives the TDI of 0.175 mg/kg bw/d [3].
 * World Health Organization (WHO)**

The EFSA suggested TDI for melamine was 0.5 mg/kg bw/d, and this number considers the effect to melamine exposure over a short period of time through migration from plastic cups or kitchenware, and also consumption of melamine contaminated product. The EFSA determined that all products from China that contain more than 15% milk are subjected to a thorough check and analysis to determine the level of melamine, and if the melamine level exceed 2.5 ppm the products will be destroyed [4]. Currently all the Chinese manufactured milk and dairy products are banned from the European Union.
 * European food safety authority (EFSA)**

The NZFSA TDI of melamine is determined by the weight and the daily intake of the person: the weight of the person is multiplied by 0.5mg (ex. a 20kg child will be 20*0.5=10mg) [11], and decided that all food should not contain more than 5ppm of melamine. For infant formula, it must not exceed 1ppm. For all other dairy-based food the melamine level should not exceed 2.5ppm. This is because foods that contain low level of milk such as candies and milk might contain melamine, however they are usually consumed infrequently and in small amount, hence they are not considered to be high risk food [4].
 * New Zealand food safety authority (NZFSA)**

According to a new study, the current FDA determined TDI value, 0.063 mg/kg bw/d is not conservative enough because the 63 mg/kg observed to be the NOAEL could be actually the lowest-observed-adverse-effect level (LOAEL) [3]. Using a different method, known as the benchmark does approach, the suggested TDI is calculated to be 0.00809 mg/kg bw for adult and less than 0.3 ppm for infants [3].

Detection Methods
Before the melamine contamination incident, food products have not been tested for melamine except for plastic safety (migration from plastic wrappers or cups) or insecticide residue [1]. However after the melamine contamination incident many new detection methods were invented to prevent further contamination.

The GC/MS method is one of the most common methods used to analyze samples for melamine. The GC/MS methods can be used to analyze both solid food sample and liquid food sample. For solid food analysis such as barley, sorghum, maize, gluten and animal feed [14], the sample can be prepared by solid phase extraction (SPE), then run in the GC/MS[14][17]. For melamine analysis, the DB-5MS column is generally used. However, due to the strong polarity of melamine, such column will give very low sensitivity. For a strong compound such as melamine a strong polar column such as the polyethylene glycol column should be used, but this kind of column is not suitable for the GC/MS since it will cause column bleeding [15]. A new method had been developed by Xiao-min Xu et al. [15]. In this method the DB-5MS column is coupled with the strong polar column Innowax (polyethylene glycol) by a quartz capillary column connector. Although there will still be band broadening because melamine is only compatible to the Innowax column, however it is shown that the efficiency of the coupled column is enhanced by 290 times[15]. Although the column bleeding still occurs 4 times more than using the DB-5MS column, it is still 1/10 times less than using just the Innowax column [15]. Compared to the LC/MS/MS method, the GC/MS method is relatively cheaper, and also easier to maintenance.
 * Gas Chromatography/Mass Spectrometry (GC/MS)**

The HPLC/MS method is the most compatible instrument for melamine detection in food and beverage samples because it is fast and sensitive. However the ionic character of melamine makes it difficult to obtain a good retention time in a C18 column. A method developed by Maria Ibanez et al. [16], by adding an ion-pair reagent such as heptafluorobutyris (HFBA) or tridecafluoroheptanoic (TFHA) to both the mobile phase and the sample, it should prolong the retention time of melamine in the column. In the study, although the HFBA did not give a satisfactory retention time of melamine, the TFHA gave a satisfactory chromatographic behavior. By adding 10mm of TFHA to the sample vial and 0.5mm of TFHA to the mobile phase [16], the result gives both a good retention time and good peak shape in samples. For solid food sample such as egg, an Ultra-Performance Liquid Chromatography tandem mass spectrometry (UPLC-MS/MS) was developed by Xi Xia et al. [17], and the sample has to be prepared by SPE.
 * High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS)**

ELISA is one of the cheaper methods for melamine determination in milk, and compared to other chromatographic methods, ELISA is much less laborious. There are specific melamine ELISA kits for melamine analysis, and this assay only takes about an hour with high sensitivity [23]. Most ELISA kits uses a similar operation principle, with melamine antibody coated microplates, horseradish peroxidase (HRP) conjugated melamine and chromogenic HRP substrate. Both the unknown sample and the HRP conjugated melamine are added to the antibody coated microplates. Base on the competition principle [25], the binding ratio is equal to the concentration ratio; hence the amount of bound HRP conjugated melamine is dependent on the amount of free melamine. The unbound material is then removed by the microplate washer, then the HRP substrate proportional to the HRP conjugated melamine is added. When the HRP enzyme reaction is finished after the incubation time the color intensity from the color dyed is measured using a microplate photometer. The concentration of melamine can then be measured by using the software included with the ELISA instrument and the calibration curve method.
 * Enzyme-linked immunosorbent assay (ELISA)**

Raman spectroscopy is probably the quickest and the easiest method for melamine detection, as it does not require any sample preparation at all; the powered milk sample can be placed into a vial and it is ready to be scanned. This method is very useful for quick onsite inspection, since there are new portable Raman instrument such as the Inspector Raman TM 1.3 (Delta Nu Inc., Laramie, Wyoming, USA) [21]. This instrument only requires a laptop computer with the compatible software, which then the data can be analyzed. The laser Raman spectrometry is much more practical compare to any other methods for onsite testing, which also gives good quantitative and qualitative characterization [21].
 * Laser Raman Spectrometry**

The Mannich reaction involves an amino alkylation of an acidic proton placed next to a carbonyl functional group with formaldehyde and ammonia or any primary or secondary amine [18][19][20]. For the study by Jamil Rima et al. [18], they hypothesized that the reaction between uranine, formaldehyde and melamine is similar to the Mannich reaction. Since formaldehyde does not have a UV spectrum, the melamine compound and the formation of a complex via the Mannich reaction will result in a peak in the UV spectrum, and the sample can be quantitatively analyzed for the melamine content by calibration curves methods. This method is much simpler compared to LC/MS/MS and the GC/MS methods, however the limitation of this method is the power of the spectrophotometer. Any food sample such as milk, pet food and wheat gluten can be used, and even complex matrix such as fish can also be analyzed [18].
 * UV Spectroscopy and the Mannich Reaction** [18]

The CZE and SPE method developed by Liang Meng et al. [22] is a new method that yields good recovery (94 to 102%) and good RSD (1.5-4.1%) [22]. This method can analyze both liquid and solid food samples, the samples were prepared by first adding sodium deoxycholate to precipitate the protein content in the sample, and then perform a SPE to clean up the sample. The sample can then be analyzed by CZE-UV. There are many other methods that were developed, however some of them is in another language other than English which makes it difficult to understand and follow. For instance, a method that involves in adding metal to liquid milk to test for the melamine content is developed by Huanwang Jing et al. [25]. In his article he stated that this method is quick and much more affordable compared to the LC/MS/MS or GC/MS methods. There isalso another method developed by Mizuka Fujita et al. [24] using the HPLC methods to analyze various Chinese processed food sample such as pork bun and crepe. If these articles can be properly translated it would be benefit to the international community.
 * Capillary Zone Electrophoresis with Solid Phase Extraction (CZE and SPE)** [22]