Melamine is an organic base and a trimer of cyanamide, with a 1,3,5-triazineskeleton. Like cyanamide, it contains 66% nitrogen by mass and, if mixed with resins, has fire retardant properties due to its release of nitrogen gas when burned or charred, and has several other industrial uses. Melamine is also a metabolite of cyromazine, a pesticide. It is formed in the body of mammals who have ingested cyromazine. It has been reported that cyromazine can also be converted to melamine in plants.

Melamine combines with cyanuric acid and related compounds to form melamine cyanurate and related crystal structures, which have been implicated as contaminants or biomarkers in protein adulterations.



Milk, infant formula and other dairy products were recently found contaminated with melamine to increase the apparent protein content, following an earlier melamine contamination outbreak in pet food in 2007.

Toxicology studies show ingestion of melamine in large quantities may lead to reproductive damage or bladder cancer due to the formation of bladder/kidney stones. At lower levels, melamine and cyanuric acid are absorbed into the bloodstream. Together, they concentrate and form melamine cyanurate in the urine-filled renal microtubules. Crystallization blocks and damages the renal cells that line the tubes, causing the kidneys to malfunction.

After 2007 and more recent melamine contamination outbreaks, there is an urgent need for analytical methods that can identify and quantify melamine in food. Current melamine detection methods involve LC-MS and GC-MS. GC-MS methods require derivatization, and LC-MS methods generally use gradient conditions that require column clean up and re-equilibration.

However, the high cost of operation and maintenance of GC/LC-MS systems as well as the labor intensive derivatization that GC-MS requires limits their use especially in the milk product factories. The HPLC-UV method therefore is presently the popular choice for most factories. In this method, melamine is separated on a C8 or C18 column using an ion pair buffer (mixture of citric acid and sodium 1-octane sulfonate) and acetonitrile mobile phase.

Here we determined melamine in milk powder samples following the regulated method.  Melamine was separated from other components in the powdered milk samples using a Brownlee Analytical C8 column and a Perkin Elmer 200 series HPLC system with UV detection. The results of method detection limits (MDL), recovery, and permitted detection deviation match the requirements in the regulated method.

We also tried a method of quantification in higher concentration (% level) in solid form using Perkin Elmer Spectrum 100 FTIR.


This writeup describes the analytical method used for the extraction and quantitation of melamine in liquid and powdered infant formula. It may also be used to extract melamine in milk, yogurt beverages and various products containing milk powder.

The method uses liquid/liquid extraction and cation exchange solid phase extraction to prepare samples, and high performance liquid chromatography with UV detection to quantitate melamine.


Water form Merck

Methanol (CH3OH) HPLC grade from Merck

Acetonitrile (CH3CN) HPLC grade from Merck

Trichloroacetic acid , analytical grade from Merck

Sodium 1-heptane sulphonic acid (98%)

Melamine (99%), HPLC grade from Merck


Analytical Column: Brownlee Analytical C8, 5 μm, 4.6 × 250 mm

Mobile Phase: Buffer (citric acid and sodium 1-heptane sulfonate : CH3CN (85:15, v/v)

Column Temp.: 40 °C

Flow Rate: 1.0 mL/min

Inj. Volume: 20 μL

UV Detection: Absorbance at 240 nm


Nine working standard solutions were prepared for calibration by adding defined volumes of stock standard solution (1000 μg/mL) and diluting with mobile phase. The concentrations of melamine solutions are 0.2, 0.5, 1.0, 2.0 and 20.0, 40.0, 60.0, 80.0, 100.0 μg/ml.


Recovery Study

2g of milk powder samples were spiked with 3 different concentrations of Melamine followed by cleanup through Silica column and the recovery data are given below:

Sample No. Conc. Of melamine spiked Conc. Of melamine obtained Recovery (%)
1. 20 mg/ml 19.2 mg/ml 96.0
2. 10 mg/ml 9.46 mg/ml 94.6
3. 2 mg/ml 1.8 mg/ml 90.0

Method Performance

Method in this application note Requirement in the regulated method
Correlation Coefficient 0.998218 (20 – 100 mg/ml) 0.999522 (0.2 – 2.0 mg/ml)  
Linearity range (mg/ml) 0.2 – 100 0.8 – 80
Recovery (%) 93.5 80 – 100


Since chromatography analysis requires solvents of high purity, a tedious sample cleanup step, we have tried to analyze Melamine by a further easier and simple technique i.e. FTIR using HATR for detection of melamine in higher concentration level (in % level).

Standard Melamine and KBr (IR grade) are oven dried and desiccated.  Three standard samples were prepared by dilution with KBr, 25%, 50% & 75%. A standard curve has been drawn which shows a good correlation.


Recovery Study using FTIR

Three synthetic samples were prepared containing 20%, 40%, 60% of melamine and quantified against the standard curve drawn. The obtained data are given below:

Sample No. Conc. Of melamine added Conc. Of melamine obtained Recovery (%)
1. 30% 29.2% 97.3
2. 40% 39.0% 97.5
3. 60% 59.1% 98.5

Method Performance

Method in this application note
Correlation Coefficient 0.9911
Linearity range (%) 25 – 75%
Avg. Recovery (%) 97.76%



As observed, preferred method for detecting Melamine in extremely low concentration is by HPLC. But in order to check the purity of the Raw Material, FTIR method with HATR is preferable.

Contributed by : Ms. Nandita Das,  Mr. Bamdev Sinha under guidance of Prof. Barun Gupta


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