Bulletin UASVM, Veterinary Medicine 67(2)/2010 ISSN 1843-5270; Electronic ISSN 1843-5378
Determination of Melamine Residue in Food Using Gas Chromatography Tandem Mass Spectrometry Iuliana GÂJÂILĂ1, C-tin SAVU1, Marinela IVĂNESCU2, Magdalena LIONIDE2, O. SAVU1, G. GÂJÂILĂ1, G. COTOR1, Mimi DOBREA1 1
University of Agricultural Sciences and Veterinary Medicine Bucharest, Faculty of Veterinary Medicine, Splaiul Independentei 105, 5th district 2 Institute for Hygiene and Veterinary Public Health Bucharest, 2th district
[email protected] Abstract. In the last two years melamine has become matter of great interest. This substance has been abused during food and feed adulteration by increasing the content of nitrogen compounds in these products. Individually, melamine is not toxic. When combined melamine and cyanuric acid in the bloodstream, they accumulate in the kidneys and form very large insoluble crystals (melaminecyanurate), which in turn can lead to renal failure and ultimately, death. In this paper we present analytical method for melamine in different food matrices by gas chromatography with tandem mass spectrometry. Keywords: melamine; food; GC-MS/MS
INTRODUCTION Melamine (2,4,6-triamino-1,3,5-triazine) is an organic base chemical most commonly found in the form of white crystals rich in nitrogen. Melamine is produced in large amounts, primarily for use in the synthesis of melamine–formaldehyde resins for the manufacture of laminates, plastics, coatings, filters, glues or adhesives, and dishware and kitchenware, but it is not approved as an ingredient in food. Analogues (cyanuric acid, ammeline and ammelide) can be produced as impurities during the manufacturing process for melamine. Melamine and its analogues are not approved as ingredients in food. However, due to the high nitrogen content (66% by mass), melamine is deliberately added to food or foodrelated products because protein content is usually estimated by determining the nitrogen content, and the added melamine can boost the nitrogen content of the products so as to make them appear to have more protein and to reduce the costs. As a pure substance, melamine in small doses is practically non-toxic. Melamine is not metabolized by animals and is rapidly eliminated in the urine. Even though the acute toxicity of melamine is low, little is known about its side-products and possible interactions amongst them. Melamine can form self-associating, high molecular weight complexes through intramolecular networks of hydrogen bonds and π-π aromatic ring stacking interactions with cyanuric acid and other analogues, as well as with uric acid and other cyclic imide-containing biomolecules. The main toxic effects in experimental animals tend to be urolithiasis or bladder stone development. This toxicity has prompted the FDA to set a standard for human consumption at 0.063 mg per kg of body mass per day. In March 2007, public awareness of melamine contamination was heightened when the contaminant was found in pet food ingredients imported from China, causing the death of
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many animals. Following this scare, it was revealed that melamine-tainted fodder may have been used to feed animals, including chickens, swine and catfish intended for human consumption. In 2008, high levels of melamine were detected in some infant formula and other liquid and powdered milk products originating from China. There were more than 52 thousand cases reported in China with a diagnosis of kidney disease. Melamine was intentionally added to milk to adulterate higher protein content in milk after dilution with water. In addition to infant food, melamine was found in many milk products. The discovery of melamine in pet food, animal feed, and protein sources including wheat gluten, rice protein concentrate, and corn gluten created an urgent need for rapid methods for detecting melamine in food. The main analytical methods available for melamine quantification involve GC-MS, HPLC and LC-MS. An HPLC method can be used to quantitatively analyze melamine at ppm level. But it is inadequate for qualitative analysis and trace-level analysis. LC-MS is a sensitive technique for quantitative and qualitative analysis. However, due to the high price of equipment, it is not widely used in practical applications. GC-MS is an economic and commonly used technique for most of analytical laboratories. MATERIAL AND METHODS Materials: different food categories analyzed in 2010 at the Institute for Hygiene and Veterinary Public Health Bucharest. Analysis method: gas chromatography triple quadrupole tandem mass spectrometry (GCMS/MS) is method for low level quantification of melamine. The limit of quantification is 0.15 mg/kg. Method requires derivatization of the small polar, and therefore relatively involatile, melamine analytes, which is followed by the analysis of the compounds in a single injection. Trimethylsilyl derivatives are analytes in the electron impact ionization mode. Samples are extracted using a mixture of acetonitrile/water/diethylamine and the analytes are subsequently converted to trimethylsilyl derivatives for analysis. Extraction: a 0.5-g sample is sonicated in 20 mL of 10:40:50 (by volume) solution of diethylamine/water/acetonitrile. The sonication period is 30 minutes to allow for complete extraction. Following sonication, the sample is centrifuged for 10 minutes at 4500 rpm to settle fine particulates from the sample matrix. The supernatant fluid is filtered through a 0.45 μm nylon filter. Once filtered, 200 μL of the solution is evaporated to dryness at 60°C with a flow of clean, dry nitrogen gas. Once dry, the sample is ready for derivatization. Derivatization: the dry sample is reconstituted in an autosampler vial with 200 μL of pyridine and 100 μL of a 0.5 μg/mL internal standard solution. Melamine and related compounds are converted to trimethylsilyl derivatives with the reagent for derivatization consisting of bis (trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS); 200 μL of this solution is added and the sample is incubated at 70°C for 45 minutes. The prepared sample is injected in the gas chromatograph coupled with mass detector. The GC-MS/MS system used in this paper was the Varian CP 3800. Parameters for Selected Ion Monitoring - Multiple Reaction Monitoring: m/z 171, 327, 342.
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Table 1 Criteria for confirmation Compound Melamine
Ions Monitored 171, 197, 327(+), 324 (+) = base ion
Relative retention time (min) 16.013
Ions 171/327 197/327 342/327
Relative ratio 0,629 0,135 0,608
RESULTS AND DISCUSSIONS Results of determinations of melamine residues in the samples analyzed are presented in Table 2. Table 2 Results of melamine residues determination by GC-MS/MS Food category
No. of samples
Range (mg/kg)
No. of ND* (%)
Upper bound (mg/kg)
102 (82.3) 5 (100)
Lower bound (mg/kg) 0.052 -
All products tested Infant formula/ baby food Powdered milk Biscuits, cakes and patiseries Candy and chocolate Soy drinks Soya powders, proteins and meals Other powdered ingredients (aa, etc.) Coffee, coffee drinks and milk teas Ammonium bicarbonate * ND – non-detected
124 5
˂0.1-2470 ˂0.1-˂2.5
3 21
˂0.17-˂2.5 ˂0.17-˂2.5
3 (100) 17 (80.9)
0.150
0.751
29 15 26
˂2.5 ˂0.2-˂2.5
25 (86.2) 11 (73.3) 17 (65.4)
0.152 0.155 0.112
0.253 0.892 0.902
7
˂0.2-˂2.5
6 (85.7)
0.308
0.308
3
˂0.17-˂2.5
3 (100)
-
-
15
70-2470
14 (93.3)
0.534
0.534
0.902 -
CONCLUSIONS
The adulteration of milk and milk products with melamine in China has prompted investigations worldwide into the concentrations of melamine and, in some cases, related analogues in milk, milk ingredients and composited products containing milk-derived ingredients. The import of milk and milk products from China into the EU is prohibited. However, composite food products, such as biscuits and chocolate, which could be made from contaminated milk powder, may have reached the EU. As a result, targeted sampling has been conducted by many member states, including Romania.
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The presence of high levels of melamine in suspect foods other than milk-based products and products containing milk-derived ingredients, such as ammonium bicarbonate and non-dairy creamers was investigated. Analytical method using gas chromatography triple quadrupole tandem mass spectrometry method (GC-MS/MS) identifies and quantifies the melamine in milk powder and other products with high protein content at low concentrations exceeding 0.05 mg/kg, which is 50 times less than the maximum permissible EU. Technical MS/MS eliminates the matrix effect, resulting in a higher confidence in identifying the potential positive samples to quantification the analyte of interest. REFERENCES 1. Andersen WC, Turnipseed SB, Karbiwnyk CM, Clark SB, Madson MR, Gieseker CM, Miller RA, Rummel NG, Reimschuessel R (2008) Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry. J Agric Food Chem 56:4340–4347. 2. EFSA (2008a) Statement of the EFSA on risks for public health due to the presence of melamine in infant milk and other milk products in China. Parma, European Food Safety Authority, EFSA Journal, 807: 1–10. 3. FDA (2007) GC-MS Method for Screening and Confirmation of Melamine and Related Analogs. 4. Heller DN, Nochetto CB (2009) Simultaneous Determination and Confirmation of Melamine and Cyanuric Acid in Animal Feed by Zwitterionic HILIC Chromatography and Tandem Mass Spectrometry, in press, Rapid Commun. Mass Spectrom. 5. Ibáñez M, Sancho JV, Hernández F (2009) Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography–tandem mass spectrometry. Anal Chim Acta 649:91–97. 6. Litzau JJ, Mercer GE, Mulligan KJ (2008) GC-MS screen for the presence of melamine, ammeline, ammelide, and cyanuric acid. US Food and Drug Administration Laboratory Information Bulletin No. 4423. 7. Tittlemier SA, (2010) Methods for the analysis of melamine and related compounds in foods: a review. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment, 27, 129-145. 8. Vail T, Jones PR, Sparkman OD (2008) Rapid and unambiguous identification of melamine in contaminated pet food based on mass spectrometry with four degrees of confirmation. J Anal Toxicol 31: 304–312. 9. Yang S, Ding J, Zheng J, Hu B, Li J, Chen H, Zhou Z, Qiao X (2009) Detection of melamine in milk products by surface desorption atmospheric pressure chemical ionization mass spectrometry. Anal Chem 81:2426–2436. 10. Yongliang L, Chao K, Kim MS, Tuschel D, Olkhovyk O, Priore RJ (2009) Potential of raman spectroscopy and imaging methods for rapid and routine screening of the presence of melamine in animal feed and foods. Appl Spectrosc 63:477–480.
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