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Following an earlier incident in 2007 where melamine was detected in pet food placed on the US American market, several methods based either on GC-MS or LC-MS/MS were developed and made available by US food and feed safety authorities. The WHO International Food Safety Authorities Network (INFOSAN) has also listed and commented on various analytical methods available for melamine detection.
None of these methods have been evaluated by collaborative study, and the matrices for which these methods were developed and in-house validated are animal tissue, wet and dry pet food, and protein concentrates of vegetable origin (gluten, soya protein). However, these methods may also be adapted for testing dairy products and complex food containing dairy ingredients. The following text summarises the methods, and more details can be obtained by following the links.
Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS; US Food and Drug Administration, Center for Food Safety and Applied Nutrition
This method consists of an initial extraction with 2.5% aqueous formic acid, followed by a series of filtration, centrifugation, and dilution steps. Both compounds are analyzed in the same chromatographic program using a zwitterionic HILIC LC column. Electrospray ionization is used in both the negative ion (CYA) and positive ion (MEL) modes. Two selected reaction monitoring (SRM) transitions are monitored for both compounds. The amount of compounds present is determined with a calibration curve consisting of sample extracts from infant formula fortified from 0.25 to 5 µg/g that have been taken through the extraction procedure. The range of recovery from fortified infant samples (n =38) was 70-114 % (RSDs 4.5- 22.7 %), and 72-110% (RSDs 5.7-24.9%) for cyanuric acid and melamine, respectively. The limits of quantification and confirmation are 0.25 µg/g for both analytes in dry infant formula.
Interim Method for Determination of Melamine and Cyanuric Acid Residues In Foods using LC-MS/MS: Version 1.0; Center for Food Safety and Applied Nutrition
In this procedure, both cyanuric acid and melamine are extracted from tissue and infant formula with a 50-50 acetonitrile-water extraction solution, followed by centrifugation. The cleanup procedures for melamine involve mix mode cation exchange (MCX) solid phase extraction (SPE) and that for cyanuric acid uses mix mode anion exchange (MAX) SPE. Each compound is analyzed separately using a zwitterionic HILIC LC column. Electrospray ionization is used in both the negative ion (cyanuric acid) and positive ion (melamine) modes. Commercially available, isotopically labelled internal standards for each compound were used to correct for any matrix effects. The method limit of quantitation (LOQ) for melamine was: 25 µg/kg for tissue and liquid formula and 200 µg/kg for dry infant formula powder. The method LOQ for cyanuric acid was: 50 µg/kg for tissue and liquid formula and 200 µg/kg for dry infant formula powder. Fortified test portions were within 75-125% recovery.
GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide and Cyanuric Acid (Version 2.1); US Food and Drug Administration, Center for Veterinary Medicine
The procedure was developed to screen various matrices for the presence of melamine and some related compounds at the established minimum reporting level (MRL) of 10 µg/g and above using gas chromatography/mass spectrometry. Samples are extracted using a mixture of acetonitrile/water/diethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis.
The procedure has been evaluated using dry protein materials (wheat gluten, rice protein, corn gluten, and soy protein), wet and dry pet foods, and dry animal feeds. It is anticipated that the method will also be applicable to a variety of other matrices.
Recently, the Belgian Federal Agency for the Safety of the Food Chain has validated this method for matrices such a milk powder, candy and biscuits.
LC/MS/MS Screen for the Presence of Melamine in swine and poultry tissues. FERN (US Food Emergency Response Network)
Melamine is extracted from muscle tissue with a mixture of acetonitrile and water. The extract is cleaned up by liquid/liquid extraction with methylene chloride followed by solid phase extraction (SPE). Melamine is eluted from the SPE cartridge with 5% ammonium hydroxide / methanol, evaporated to dryness, reconstituted with internal standard and 50% acetonitrile / water, and analyzed by LC/MS/MS.
The method is applicable for screening of melamine in swine and poultry muscle at ≥ 50 ppb.
Determination of Melamine Residues in Catfish Tissue by Triple Quadrupole LC-MS-MS with HILIC Chromatography. US Food and Drug Administration, Center for Food Safety and Applied Nutrition
A triple quadrupole liquid chromatography tandem mass spectrometry method is presented for the quantitative determination and confirmation of melamine residues in catfish. Catfish tissue was extracted with 50:50 acetonitrile:water and 1 N hydrochloric acid and cleaned-up using solid phase extraction cartridges. Extracts were analyzed by LC-MS-MS with HILIC chromatography and electrospray ionization in positive ion mode. Catfish tissue was fortified at 10, 25, 50, 100, and 500 ng/g (ppb). The average recovery of melamine from fortified samples (n = 17) was 76.3 % with an RSD of 14.3 %.
Health Canada has modified this method for application to various milk containing food products.
Updated FCC Developmental Melamine Quantitation (HPLC-UV). US Food and Drug Administration, Center for Veterinary Medicine
This method extracts melamine from gluten or moist pet food using acetonitrile/water. Separation and quantitation is by ion-pair HPLC and a UV detector.
Linear range: established from 1.0 – 400 mg/ml; calibration range 1.0 – 200 mg/ml.
Reproducibility: duplicate preparations agree within 0.1 - 5% relative basis
Spike/recovery (based on spiking solid melamine powder into test matrix prior to extraction): 90 -110%.