Determination of melamine in feed: Validation of a gas chromatography–mass spectrometry method according to 2004/882/CE regulation
Introduction
Melamine (2,4,6-triamino-s-triazine), is a chemical intermediate used to manufacture amino resins and plastics.
This industrial chemical was responsible for the massive recall of more than 100 name brands of pet foods in the last year, following reports of pet illnesses and deaths from liver failure in the United States beginning February 2007 (Burns, 2007a, Burns, 2007b).
The US Authorities began an investigation to trace the source of these animal health problems and it was found that wheat gluten imported from China and used for the production of pet food was the origin of these problems. Further investigations revealed that melamine, an industrial chemical high in nitrogen that is used in such materials as plastics had most likely been fraudulently added to wheat gluten and other protein sources to enhance the apparent protein content of these feedstuffs. Later, melamine and cyanuric acid, a compound structurally related to melamine, was found in rice protein concentrate imported from China. Melamine has also been found in South Africa in corn gluten originating from China. As the protein concentration is measured by analysis of the total nitrogen content, the addition of melamine (C3H6N6) is to enhance the apparent protein content of wheat gluten and other protein sources. In the US, besides pet food also swine feed and livestock feed has been found contaminated to a limited extent (FDA, 2007). These levels of feed contamination were estimated by USDA to unlikely place human health at risk (USDA, 2007). Toxicology studies found melamine toxicity in mammals very low (OECD, 2002), but risk assessment of melamine is complicated by the fact that it can exert differential toxicity in various animal species (Baynes et al., 2008).
Although melamine and cyanuric acid have only been found in wheat gluten, rice protein concentrate and corn gluten used in animal feed, it cannot be excluded that melamine and structurally related compounds have been added to other protein sources intended to be used for feed or food.
Therefore, in line with the actions taken by the US authorities, and although there is no evidence that contaminated wheat gluten or rice protein concentrate or any other protein source originating from China have been imported into the EU, Member States have been asked by the Commission to control consignments of wheat gluten, corn gluten, corn meal, soy protein, rice bran and rice protein concentrate originating from third countries and in particular from China, for the presence of melamine and structurally related compounds (such as cyanuric acid, ammeline and ammelide) and to report the results of the control (both favourable and unfavourable) to the Commission through the Rapid Alert Systems for Food and Feed (RASFF).
The US Food and Drug Administration proposed a GC–MS method for the screening of melamine and related compounds which should be regarded ad interim cause the method was not validated (FDA, 2007).
Our aim is to develop and validate a quantitative method to detect melamine in dry animal feed with a limit of quantitation (LOQ) of 10 mg/kg, as previous data (FDA, 2007) showed melamine in raw materials at levels in the range of 2–80 g/kg. Then, referring to the FDA method, we validated a quantitative gas chromatography–mass spectrometry method according to 2004/882/CE regulation “on official controls performed to ensure the verification of compliance with feed and food law”.
Section snippets
Samples
Our validation study was carried out on 20 feed samples: 8 raw mat and 12 complete feed. The raw materials were barley (2), sorghum (2), maize (2), gluten (2); while the complete feed were for poultry (2), fish (4), cattle (2), swine (2) and pet (2).
Materials and reagents
Acetonitrile Pestanal Riedel-de Haën, diethylamina (DEA) Sigma-Ultra and pyridine (Fluka, absolute, over molecular sieve H2O ⩽0.005%) were purchased from Sigma (Milan, Italy).
N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (BSTFA
Quantitative method development
The method allows for the unequivocal identification of melamine, as shown in Fig. 1, Fig. 2. The negative pool chromatogram used as matrix for the calibration curve and as matrix to spike is displayed in Fig. 1.
In Fig. 2 the spiked matrix (75 mg/kg) is provided, while in Fig. 3 the mass spectrum of the derivatized standard of melamine and DACP is displayed.
In this analytical procedure DACP was used as internal standard to check the derivatization process. If DACP signal is not detected or low
Discussion and conclusion
In this study, a GC–MS method was developed, following the US Food and Drug Administration technique, to detect and quantify the presence of melamine in feed.
The complete validation of this quantitative GC–MS method is presented.
The derivatizing solvent brand may be a critical factor; the same solvent must be used for all the analytical trial (standards and samples).
The data reported showed that this method is suitable for routine analysis of melamine in feed with good recovery and with
Acknowledgements
This study has been carried out with the financial support of the Piedmont Region and the Italian Ministry of Health.
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