Melamine analysis by HILIC

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Last updated: 2009-09-16, 16:20

SeQuant® ZIC®-HILIC HPLC columns are suitable for determination of melamine and analogues in pharmaceuticals and milk products. Links to methods for analysis, guidelines from official authorities and background information are available below.

FDA Issues Pharmaceutical Industry Guidance on Preventing Melamine Contamination

August 6, 2009 the U.S. Food and Drug Administration issued a Guidance for Industry - Pharmaceutical Components at Risk for Melamine Contamination [pdf]. It says that certain pharmaceutical ingredients used in the manufacture or preparation of drug products are recommended to be screened for melamine, see list.


"The FDA urges implementation of appropriate controls to assure consumers that melamine contamination will not happen in the pharmaceutical supply chain," said Commissioner of Food and Drugs Margaret A. Hamburg, M.D. "We look forward to working with industry on this serious public health concern."


At the moment, FDA has no reason to believe that pharmaceutical supplies are contaminated with melamine, but the recent events involving pet and livestock food products in the United States, and milk products for infants in China, illustrate the potential for drug components to be contaminated with melamine.


Hence, it is important for drug manufacturers to assure that no component used in the manufacture of any drug is contaminated with melamine. FDA recommends that compounders who use at-risk components in drugs ensure proper testing.


Requirements and recommendations

The guidance for pharmaceuticals recommends the use of FDA-published methods based on equipment generally available to pharmaceutical manufacturers or contract testing labs.


The test method used should be suitable to assay melamine contamination down to at least 2.5 parts per million (ppm).

Recommended methods are based on liquid chromatography triple quadrupole tandem mass spectrometry (LC-MS/MS) or gas chromatography/mass spectrometry (GC-MS). The LC MS/MS method is based on HILIC and also urge the need to prevent melamine degradation during sample handling, (see FDA methods).


The compounds at risk may be, but are not limited to:

• Adenine

• Albumin

• Amino acids derived from casein protein hydrolysates

• Ammonium salts

• Calcium pantothenate

• Caseinate or sodium caseinate

• Chlorophyllin copper complex sodium

• Colloidal oatmeal

• Copovidone

• Crospovidone

• Dihydroxyaluminum aminoacetate

• Gelatin

• Glucagon

• Guar gum





• Hyaluronidase

• Imidurea

• Lactose

• Melphalan

• Povidone

• Povidone-Iodine

• Protamine sulfate

• Protein hydrolysate (powder) for injection

• Taurine

• Thioguanine

• Urea

• Wheat bran

• Zein

This list was based on the FDA Inactive Ingredient Database (IID), and is not considered to be exhaustive, and it is essential that manufacturers evaluate their drug components to determine whether they are vulnerable to melamine contamination.

Strategies for Determination of Melamine by HILIC

Melamine is a small polar compound which is very rich in nitrogen (67% by mass). It has recently been found in milk products and animal feed, where it possibly have been added to give a false impression of high protein content. It is believed that melamine combined with cyanuric acid can cause fatal kidney stones due to the formation of an insoluble melamine-cyanurate complex. Determination of melamine and other small nitrogen-rich compounds, is therefore of large importance to ensure food safety. 

Determination of melamine

Currently there are several analysis methods from different authorities around the world where the SeQuant ZIC®-HILIC HPLC column is a key part in the determination.


The Food and Drug Administration in USA (FDA) approached the problem by developing a ZIC®-HILIC, LC/MS/MS method for simulataneous determination of melamine and cyanuric acid in a single run after a simple extraction and filtration sample pretreatment [1-2]. The method has been published in the scientific journal Rapid Communication in Mass Spectrometry [1]. Merck SeQuant have summarized these findings, including complete experimental conditions, in a Technical Note [2], that can be downloaded as a pdf. FDA have continued the study and published a fully validated method for determination of melamine and cyanuric acid in infant formula [3]. 


The Ministry of Agriculture in China uses separate methods for determination of melamine and cyanuric acid [4]. Both are using different solid phase enrichment schemes, followed by ZIC®-HILIC and LC/MS/MS analysis. The methods were developed by Prof. Biying Chang and Dr. Qingsheng Liu at the Food Institute and Dr. Linyun Li at the Vegetable Institute. Prof. Biying Chang can supply more information and details about this method.


There also exists another (non-validated) FDA method for analysis of melamine and cyanuric acid in food, which uses solid phase extraction similar to the Chinese strategy above and ZIC®-HILIC, LC/MS/MS for the separation and detection [5]. The Japanese Ministry of Health, Labor and Welfare has also issued a method for melamine [6] based on sample pre-treatment and enrichment on ion-exchanger cartridges and separation on ZIC®-HILIC and LC/MS/MS analysis.


Scientific references


1. Simultaneous determination and confirmation of melamine and cyanuric acid in animal feed by zwitterionic hydrophilic interaction

chromatography and tandem mass spectrometry.

David N. Heller and Cristina B. Nochetto

Rapid. Commun. Mass Spectrom, 22 (2008) 3624-3632


2. Merck SeQuant Technical Note TN-008:

Simultaneous Determination of Melamine and Cyanuric Acid in Animal Feed via ZIC®-HILIC Chromatography - Mass Spectrometry


3. Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS

Sherri Turnipseed, Christine Casey, Cristina Nochetto, David N. Heller

FDA Laboratory Information Bulletin, LIB No. 4421, Vol 24, Oct 2008


4. Determination of Melamine and Cyanuric Acid by Zwitterionic HILIC-MS-MS

Biying Chang, Qingsheng Liu, Linyun Li

Please contact Prof. Biying Chang for more information.


5. Interim Method for Determination of Melamine and Cyanuric Acid Residues in Foods using LC-MS/MS: Version 1.0

Michael Smoker, Alexander J. Krynitsky

FDA Laboratory Information Bulletin, LIB No. 4422, Vol 24, Oct 2008


6. Determination of Melamine by solid phase extraction and enrichment, and LC/MS/MS

Japanese Ministry of Health, Labor and Welfare


7. Evaluation of the Renal Effects of Experimental Feeding of Melamine and Cyanuric Acid to Fish and Pigs

Reimschuessel R, Gieseker CM, Miller RA, Ward J, Boehmer J, Rummel N, Heller DN, Nochetto C, de Alwis GK, Bataller N, Andersen WC, Turnipseed SB, Karbiwnyk CM, Satzger RD, Crowe JB, Wilber NR, Reinhard MK, Roberts JF, Witkowski MR

Am. J. Vet. Res., 69 (2008) 1217-1228


8. Clinicopathologic, Histologic, and Toxicologic Findings in 70 Cats Inadvertently Exposed to Pet Food Contaminated with Melamine and Cyanuric Acid

Cianciolo RE, Bischoff K, Ebel JG, Van Winkle TJ, Goldstein RE, Serfilippi LM

J. Am. Vet. Med. Assoc., 233 (2008) 729-737


Health effects of melamine and cyanuric acid

• U.S. Food and Drug Administration (FDA)

Interim Safety and Risk Assessment of Melamine and Melamine and its Analogues in Food for Humans [2008-10-03]


• World Health Organisation (WHO)

New guidance issued on melamine levels in food [2008-09-29]


• European Food Safety Agency (EFSA)

EFSA assesses possible risks related to melamine in composite foods from China [2008-09-25]


FDA News

• "Analytical Methods for Melamine and Triazine Analogs" [2009-08-06]

• Food Safety [2009-07-31]

• "Melamine contamination in China" [2009-01-05]

• FDA Detects Melamine Contamination in Flavored Drink [2008-10-06]

• FDA Issues Interim Safety and Risk Assessment of Melamine and Melamine-related Compounds in Food [2008-10-03]

• FDA Updates Health Information Advisory on Melamine Contamination [2008-09-26]


More information

• Merck Chemicals page for Melamine Analysis

Wikipedia: Melamine

Wikipedia: Cyanuric acid

Wikipedia: Protein adulteration in China

Wikipedia: 2008 Chinese Milk Scandal

Wikipedia: 2007 Pet Food Recalls