Effects Of Melamine In Food Biology Essay

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For almost ten years or more, a chemical called melamine has been made popular with shocking stories. In 2007, it was reported that pet food ingredients were adulterated with melamine in the United States and Canada. Later in 2008, melamine drew most international attention when its presence in infant formula poisoned tens of thousands of babies in china. It is commonly used in the production of melamine resin that forms a very durable thermoset plastic. It is also used in making fertilizers and present in plastic ware.

Melamine is a polar organic compound with the chemical formula C3H6N6. Its IUPAC name is 1, 3, 5-triazine-2, 4, 6-triamine, and is only slightly soluble in water.

Figure1. Structure of Melamine

Effects of Melamine in Food

Melamine contains 66% nitrogen by mass, the reason why it was used as an additive to falsely increase the detected protein level above the expected level. This fraudulent addition resulted to the death of about 13,000 children which were inflicted with kidney stones and other renal complications owing to ingestion of foodstuffs adulterated with melamine. (1,2).

In November 2008, melamine in infant formula was set at zero-tolerant level by US FDA and in other foods; a maximum tolerance level was set to be 2.5 ppm. The US FDA however, put the limit at which presence of melamine infant formula present no public health concerns below 1ppm. (3,4).

A report by the World Health Organization (WHO) in October 2008, said that melamine combined with cynuric acid was also found in infant formula and formed crystals which eventually resulted into kidney stone. Whether the cyanuric acid which plays the same role as melamine in infant formula was added intentionally is not yet determined by WHO.

The continuous rise in global concern on melamine food adulteration resulted in developing different methods for its analysis in foods and dairy products.

Selective Analytical Approaches

Since the outbreak of melamine contamination in pet food, the US FDA has published six methods for melamine analysis, utilizing high performance liquid chromatography coupled with ultra-violet (UV) detector (HPLC-UV; US FDA Forensic Chemistry Center HPLC-UV method), gas chromatography coupled with mass spectrometer (GCMS or GCMS/MS; US FDA Laboratory Information Bulletin 4423), and liquid chromatography coupled with mass spectrometer (LC-MS/ MS; US FDA Laboratory Information Bulletin 4396, 4421 and 4422). Manufacturers have also developed enzyme linked immuno-sorbent assay (ELISA) kits for detection of melamine using antigen-antibody reaction. Tandem mass spectrometry (MS/MS) provides the highest degree of selectivity, followed by single-stage mass spectrometry (MS), diode array detection (DAD) and, lastly, ultraviolet absorption (UV).

The complex milk matrix

Milk, the primary source of dairy is an essential component of human diet. Apart from water, which takes largest percentage of milk (88%), other nutrients such as proteins, fat and sugars, all of which are completely solubilised are also found in milk.

Minerals and vitamins such as calcium and potassium are also present in milk. All these make milk a complex matrix and difficult to analyse.

Sample preparation

Selective analytical methods adopt relatively simple procedures for sample preparation. This involves liquid extraction followed by further clean-up with solid-phase extraction (SPE). Clean-up is critical owing to the complexity of milk the major component of dairy products. If the detection limits of the adopted method are low, more intensive clean-up procedures are needed. Because of its high specificity and selectivity, mass spectrometry detection is typically used for the analysis of melamine. The major impediment is the complex matrices of the analytes of interest, the reason why the sample is required to be clean-up. In general, the sample preparation procedures employed are interchangeable with the various detection methods.

Owing to the polar nature of melamine sample extraction is carried out with polar solvents. Example of such includes formic acid, acetic acid, hydrochloric acid, phosphate buffer at pH 5, acrylonitrile, and methanol.

Melamine bonded with cyanuric acid in the sample leads to formation of supramolecular aggregates that are insoluble in water. However, Filigenzi et al stated that solutions of diethylamine/acrylonitrile/ water effectively dissolve melamine cyanuric acid complex at concentrations up to 10 mg/10 ml (2008). Also, a report by FDA suggested that exposing the samples to very high pH (basic or alkaline) or very low pH (acidic) with sample dilution is an effective means for disrupting this complex, thereby aiding solubility.

Sample cleanup processes

Sample clean-up can be carried out in two major ways. The first one involves exposing the milk sample to 2.5% formic acid, a low pH condition followed by further exposure to acetonitrile, an organic solvent to allow precipitation of protein. The precipitate formed is removed by centrifugation. It was reported that this approach called dilute-and-shoot technique is suitable for fast screening of samples for melamine.

The second cleanup approach involves the use of protein precipitation step (similar to the dilute-and shoot technique) followed by solid phase extraction (SPE) step. This approach, according to a report is used when a better cleanup protocol is required, such as when the amount of melamine needs to be quantified accurately.

The main purpose of sample clean-up process using SPE is to reduce to the barest minimum, the impurities co-extracted with melamine and to increase the selectivity of melamine from the sample matrix. Strong cation exchange SPE or a mixed-mode SPE that includes a strong cation exchanger is normally used in this procedure. The SPE packing retains the basic melamine while the impurities are removed during the washing process. Melamine is subsequently eluted from the SPE cartridge using a strong base in methanol. Both polymer-based and silica-based SPE can be used.

Instrumental analysis

Due to small and polar nature of melamine most methods use HPLC. Recently, hydrophilic interaction liquid chromatography (HILIC) is used in the place of HPLC. Some use polar reversed-phase columns, cyano columns, or amino columns. Other methods generally use reversed-phase (C18 or C8) HPLC columns. Reversed-phase columns have a disadvantage of poor separation of small and very polar melamine compare to other methods. The use of ion pair reagents can improve separation on reverse-phase columns according to a report but can lead to ionization suppression in the MS source. The use of multiple columns in series can also be used to enhance separation.

Fewer methods have adopted GC. In GC, however, melamine must be derivatized to enhance its gas chromatographic properties, or detection by that method would not be possible. All current GC methods produce trimethylsilyl (TMS) derivatives for analysis.

Mass spectrometry detection

Because of its high selectivity, MS/MS detection method is used most often. Ability of MS/MS methods to monitor at least two ion transitions for each analyte is an added advantage. This is due to the fact that another identification criterion can be set by calculating the ratio of responses from the two transitions.

In most cases melamine is analysed together with cyanuric acid. Thus, melanine is analysed in the positive electrospray ionization mode (ESI+) and cyanuric acid in the ESI- mode in HPLC-MS/MS. The opposite polarities required necessitate the use of polarity switching during one HPLC-MS/MS run, and thus adequate analyte chromatographic separation, or two different HPLC-MS/MS runs. The transitions monitored the most often for MEL are 127 ? 85 and 127 ? 68, which correspond to a loss of cyanamide from the [M+H]+ ion to produce the 2,4-diamino-1,3-diazete cation, and subsequent loss of ammonia to form the ion with m/z = 68. The fragmentation of [M-H]- for cyanuric acid is analogous to the pathways observed for melamine. One group has used atmospheric pressure chemical ionization (APCI+) for melamine and APCI- for cyanuric acid. Derivatized melamine analytes are analysed in the positive electron impact mode (EI) in GC-MS/MS.

Single-stage GC-MS methods use EI selected ion monitoring of multiple ions to quantify the trimethylsilyl (TMS) derivatives of each melamine analyte. These methods are less selective than MS/MS methods, which results in a lowered sensitivity.

The remaining methods use less selective and less sensitive UV or DAD in combination with HPLC for detection of melamine analytes. They also require more intensive method validation to ensure that there are no interferences that absorb at the wavelengths used for detection. This can be accomplished by comparison of results with more selective MS methods. The UV and DAD methods monitor wavelengths in the 200 270 nm range, most often around 214 nm.


Quantification schemes reportedly being used were external standard quantification, matrix-matched external standard quantification and internal standard quantification.

In HPLC-ESI-MS/MS analysis, the uses of matrix-matched standards together with stable isotope internal standards were made essential for the correction of matrix effects during quantification and data analysis. According to a report, pre-extraction fortification of blank matrix leads to loss of analyte during sample processing when quantified against the matrix-matched standards. It also leads to changes in the ionization in the MS source. The change in ionization can be accounted for if post-extraction-fortified matrix-matched standards are used. The only disadvantage of using post-extraction-fortified matrix-matched standards is loss of melamine analyte which cannot be accounted for. Matrix-matched standards can only be used properly if blank matrix is appreciably closed to the matrices of the samples under analysis. This allows similarities in their matrix effects.

Stable isotope internal standards are also useful to suppress matrix effects when MS methods are used.

The In GC-MS method which involves derivatization process, internal standard 2,6-diamino-4-chloropyrimidine is used to monitor the process.


Majority of reports adopt HPLC-MS/MS methods as more sensitive than others as it can generally be used to measure melamine down to the 1 10 g/kg range. UV and DAD methods are at least 1 order of magnitude less sensitive when compare with HPLC-MS/MS methods. In addition, enzyme-linked immunosorbent assay (ELISA) methods do not routinely detect concentrations in sample matrices as low as MS/MS methods do.

Advantages with HILIC

Aqueous normal phase (ANP) liquid chromatography is a very suitable detection instrument for melamine, a water soluble molecule. ANP allows the partition of a polar hydrophilic analyte between a relatively polar stationary phase and a relatively non-polar mobile phase.

Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a version of normal phase liquid chromatography. It represents one of several mechanisms at which ANP operates. Separation of polar and hydrophilic compound like melamine is quite easier with HILIC because sample derivatization or ion-pair additive in the mobile phase are eliminated. It also yields better retention of water soluble molecules when compared to reversed-phase chromatography. This longer retention time allows better separation of the analyte in the presence of interfering species.

If these interfering components elute at the start of the chromatogram, melamine retention can be changed to ensure that it elutes away from these interfering peaks.

In contrast to reversed-phase LC, if the melamine and interfering components elute close to the start of the chromatogram, there is minimal flexibility in separating the melamine from these interfering peaks, as there is no more time or space to move either the melamine or the interfering components to a longer or shorter retention period.


There is not much information on method validation for each method used to analyse melamine in dairy products. Although, per cent recoveries of analyte from fortified samples are rarely reported within the range of 60-160% with some measure of the methods precision, these data do not give much information on each method because validation was performed on samples with little amount of matrices.

Overview of rapid screening and qualitative methods

Table 1 listed rapid screening and other qualitative methods for the analysis of melamine and structurally related compounds in foods for human consumption and animal feeds. Other methods include screening methods using ELISAs, surface-enhanced Raman spectroscopy (SERS) and time-of-flight mass spectrometry (TOFMS).

The use of TOFMS required no sample processing and had an estimated LOD of 1 mg/kg, but validation data were not provided. With this method, there is a potential for interference with melamine if the resolving power of the MS is not high enough to distinguish between the masses of the interference and melamine.