Detection of Melamine in Dairy Products
by Mass Spectrometry
Wong Ho Yi*
Department of Food and Health Sciences, Faculty of Science and Technology, Technological and Higher Education Institute of Hong Kong, China
Submission: May 25, 2020; Published: September 17, 2020
*Corresponding author: Wong Ho Yi, Department of Food and Health Sciences, Faculty of Science and Technology, Technological and Higher Education Institute of Hong Kong, Hong Kong Special Administrative Region, China
How to cite this article: Wong H Y. Detection of Melamine in Dairy Products by Mass Spectrometry. Nutri Food Sci Int J. 2020. 10(3): 555788. DOI: 10.19080/NFSIJ.2020.10.555788.
Melamine is a nitrogen-rich chemical compound and is illegally added to dairy products by manufacturers to boost the nitrogen content misleading the protein content result of Kjeldahl method (i.e. the internationally recognized method for estimating the protein content in foods). According to U.S. Food and Drug Administration (FDA), when melamine and cyanuric acid are absorbed into the bloodstream, then concentrated and interacted in the renal microtubules to form large amount of crystals (kidney stones) which block and damage the renal cells, causing kidneys damage. In 2008, there is a serious food safety incident related to melamine-tainted infant formula milk powder causing illnesses of about 300,000 Chinese infants and young children and six reported deaths. The melamine-tainted event leads to a need for sensitive and reliable techniques for detection and quantitation of melamine in dairy products. This article reviews the most commonly used mass spectrometric analytical technique that has been used for the analysis of melamine in dairy products.
Keywords: Dairy products; Melamine; Liquid chromatography; Mass spectrometry
Melamine (IUPAC name: 1,3,5-triazine-2,4,6-triamine) is an organic molecule with a molecular formula C3H6N6 and molar mass 126.12 g/mol. Chemical structure of melamine is shown in Figure 1. It is a white solid at room temperature and is an organic base in nature. Melamine has a wide range of applications, it is used to manufacture many different useful commercial products, such as tableware, polymeric cleaning foam sponge, colorant inks and fire-retardant additives. However, some manufacturers illegally added melamine to food products. Melamine-contaminated pet feed causing over thousands of dogs and cats dead from kidney failure in North America in 2007. And after one year, melamine was illegally added to infant formula milk powder causing illnesses and dead of infant and children in China in 2008. (Figure 1)
The regulation of melamine in food is that World Health
Organization (WHO) set the tolerable daily intake (TDI) for
melamine at 0.2 mg/kg of body weight per day. The Chinese
Administration of Quality Supervision, Inspection and Quarantine
has reported that the melamine content in dairy products
determined was up to 6200 mg/kg which is over thirty thousand
times of the melamine limit set by WHO . Dairy products are
food products that produced from milk. There are various dairy
products in the market, such as milk, powdered milk, infant
formula milk powder, ice-cream, butter, cheese and yogurt.
According to United States Food and Drug Administration ,
the quality control methods for dairy products involve three
major tests: Salmonella test, phosphatase test and penicillin test.
Negative results must be obtained from these tests to ensure
the dairy products are absence of Salmonella, phosphatase and
penicillin. Salmonella is bacteria that can cause typhoid fever,
paratyphoid fever, foodborne illness and human death .
Phosphatase is used as an indicator in dairy products because
the destruction of phosphatase in dairy products coincides with
the heat treatment used to kill Mycobacterium tuberculosis and
other pathogenic organisms . While penicillin is a group of
antibiotics derived from penicillium fungi, it can cause penicillin
allergy to human such as anaphylaxis, hives, below-the-skin
swelling and asthma symptoms . However, melamine test does
not involve in the quality control methods for dairy products.
The purpose of illegally adding melamine to dairy products by
manufacturer is to elevate the apparent nitrogen content to give
a false protein content result of Kjeldahl test method. There are
various methods available for the detection of melamine in dairy
products, for examples, micellar electrokinetic chromatography
(MEKC), enzyme-linked immunosorbent assay (ELISA), liquid
chromatography with photodiode-array detection (LC-PAD), gas
chromatography with mass spectrometry (GC-MS) and liquid
chromatography with mass spectrometry (LC-MS) .
Mass spectrometric methods are widely used to analyze
melamine in dairy products in the world. It is because it can
provide high sensitivity and high resolution. Mass spectrometry
is a powerful analytical technique that analyze sample by ionizing
the sample and detecting the ions according to their mass-tocharge
ratios [7,8]. The molecular mass, molecular formula and
concentration of sample can be determined from the mass spectrum
obtained by mass spectrometry. It is excellent for both qualitative
and quantitative analysis. Moreover, mass spectrometer is usually
coupled with liquid or gas chromatography, LC-MS or GC-MS to
analyze sample mixture. The mass spectrometer acts as a detector
for the chromatography. Sample components that separated by
chromatography are then analyzed by the mass spectrometer. The
mass spectrum obtained is a plot of ion intensity against m/z ratio.
The m/z ratio of molecular ion and fragment ions can provide
structural information of the sample molecule for identification.
And the ion intensity is proportional to the concentration of the
sample molecule that can be used for quantitative analysis. The
results obtained from mass spectrometry are high in accuracy
and precision. This review is aimed to present an overview of the
detection of melamine in dairy products by mass spectrometry.
The most widely used mass spectrometric method, i.e. liquid
chromatography with mass spectrometry (LC-MS) is discussed.
In order to determine melamine in dairy products, liquid
chromatography coupled with mass spectrometry (LC-MS)
is the preferred method because this technique can give a
high selectivity, sensitivity and reproducibility. The analytical
ability of this technique can also be enhanced by using tandem
mass spectrometry MS/MS or MSn which can prevent false
positive results, and hence precise, accurate and more reliable
identification and quantification results can be obtained .
Good adaptability is also one of the advantages that using mass
spectrometry to determine melamine in dairy products because
mass spectrometry can be applied to the same analyte in different
matrices of dairy products .
Liquid chromatography with tandem mass spectrometry
(LC-MS/MS) analysis is the most commonly used method for
determination of melamine in dairy products. By using internal
standard method, melamine-13C3 and amino-15N3 labeled
melamine (its chemical structure is shown in figure 2) is used as
the internal standard to enhance the sensitivity and accuracy by
reducing errors from the matrix effect or enhancement effects
during mass analysis . The internal standard used should be
a compound with similar chemical and physical properties, but
different mass as compared to the compound to be measured. A
calibration curve is obtained by measuring samples containing the
fixed quantity of the internal standard and increasing quantities
of the melamine. The sample to be quantified is then measured
with a constant quantity of internal standard added to it. Finally,
the unknown concentration of melamine in the dairy products can
be calculated from the calibration curve. (Figure 2)
Dairy products such as infant formula milk powder is a
challenge to analysis because it is a very complicated biological
samples that composed of different nutrients, including
carbohydrates, proteins, fats, linoleic acid, vitamins A, C, D, E, K,
thiamin (B1), riboflavin (B2), B6, B12, niacin folic acid, pantothenic
acid, minerals calcium, magnesium, iron, zinc, manganese, copper,
phosphorus, iodine, sodium chloride, potassium chloride and etc
. Since dairy products are very complex matrices, it generally
needs different sample pre-treatment and cleanup procedures
before the melamine analysis . Table 1 showed that the
experimental results of LC-MS/MS method for the quantitative
determination of melamine in milk-based infant formula. The LCMS/
MS results demonstrated good recovery, relative standard
deviation and limit of detection (LOD). It is a very accurate and
precise method used to determine melamine in dairy products
Melamine is a useful ingredient that can be synthesized into
different materials for different uses. The most commonly used
melamine product in the daily life is melamine-formaldehyde
tableware which is a durable and hard thermosetting plastic with
water and acid resistant properties. However, some manufacturers
illegally added melamine to food, such as pet food, dairy products
and infant formula milk powder in order to boost the nitrogen
content misleading the protein content result of Kjeldahl method.
These kinds of illegal action cause illnesses and dead of many pets,
infant and children. Acute toxicity of melamine can cause nausea,
diarrhea and vomiting. While chronic toxicity of melamine usually
results in kidney stone formation and kidneys failure. Melamine
usually interacts with its by-product cyanuric acid to melamine
cyanurate which is more toxic than either melamine or cyanuric
acid alone causing the problem worse. The melamine-tainted
events lead to a need for sensitive and reliable techniques for
detection and quantitation of melamine in dairy products. The
testing method for determining melamine in dairy products based
on U.S. FDA LC-MS/MS method is still the most commonly used
method for melamine determination in the world until nowadays
because of its high sensitivity and high accuracy and precision.
Desmarchelier A, Cuadra MG, Delatour T, Mottier P, Agric J(2009) LC-MS/MS method for the simultaneous quantitative determination of melamine and cyanuric acid in infant formula and raw cow’s milk. Food Chem 57:7186-7193.