Validation of a method of high performance liquid chromatography with fluorescence detection for melamine determination in UHT whole bovine milk Virgínia de Lourdes Mendes Finete a, b , Marcos Martins Gouv ^ ea c , Fl  avia Ferreira de Carvalho Marques a, c , Annibal Duarte Pereira Netto a, c, * a Fundaç~ ao Oswaldo Cruz, Escola Polit ecnica de Saúde Joaquim Ven^ ancio, Av. Brasil, 4365, Manguinhos, CEP 21040-900, Rio de Janeiro, RJ, Brazil b Universidade Federal Fluminense, Programa de P os-Graduaç~ ao em Química, Instituto de Química, Outeiro de S~ ao Jo~ ao Batista. s/n, 24020-141, Centro, Niter oi, RJ, Brazil c Laborat orio de Química Analítica Fundamental e Aplicada, Departamento de Química Analítica, Instituto de Química, Universidade Federal Fluminense, Outeiro de S~ ao Jo~ ao Batista, s/n, 24020e141, Valonguinho, Centro, Niter oi, RJ, Brazil article info Article history: Received 18 October 2014 Received in revised form 2 December 2014 Accepted 3 December 2014 Available online 12 December 2014 Keywords: Melamine determination Natural fluorescence of melamine Milk adulteration UHT whole bovine milk HPLC-Fluorescence detection abstract A method of high performance liquid chromatography with fluorescence detection (HPLC-Fluo), based on previous studies of the natural fluorescence emission of melamine (250/365 nm), was developed and validated for melamine determination as an adulterant of bovine UHT whole milk. The clean up treat- ment of milk samples by deproteinization was thoroughly optimized. No matrix effect was observed and a linear range from 0.05 to 10.0 mg mL 1 was obtained, with a value of R 2 equal to 0.9998. The limits of detection and quantification of melamine were 0.0081 and 0.027 mg mL 1 respectively, or 0.023 and 0.076 mgg 1 of milk, respectively, considering the sample dilution during extraction. Extracts of milk samples fortified with melamine at three concentration levels, two of which corresponded to the levels established by WHO for melamine in foods, led to an overall mean recovery of 95.4 ± 1.2% (n ¼ 9). This recovery value satisfies the performance criteria established by the Codex Alimentarius for analytical methods suitable for determination of food residues, demonstrating the usefulness and effectiveness of the proposed method. © 2014 Published by Elsevier Ltd. 1. Introduction Melamine (1,3,5etriazinee2,4,6etriamine) (Fig. 1) is a synthetic organic compound produced from urea under heat and pressure, used in the manufacture of a variety of products, including elec- trical equipment and flame-retardants. Its reaction with formal- dehyde is primarily used for the synthesis of melamine- formaldehyde resin, a very durable thermo set plastic, used for the manufacture of laminates, plastics, commercial filters, adhe- sives, and dishware/kitchenware (FAO & WHO, 2010; U.S. International Trade Comission, 1996). Despite the many legal uses of melamine, it became a potential milk adulterant owing to its high nitrogen content (66% by weight). Therefore, the addition of melamine to milk fraudulently increases the nitrogen content of milk and its protein content as determined by the Kjeldahl method (Finete, Gouv^ ea, Marques, & Pereira Netto, 2013). The ingestion of melamine causes a variety of toxic effects, including chronic kidney inflammation, due to the formation of melamineecyanurate complex stones, and their deposition in the renal tubules (Hau, Kwan, & Li, 2009). In 2008, aiming to increase the protein content of milk, large amounts of melamine were fraudulently added to infant formula and some dairy products in China (Tyan, Yang, Jong, Wang, & Shiea, 2009; Xin & Stone, 2008). More than 294.000 children were exposed and at least six of them died after ingestion of an infant milk formula contaminated with melamine (Ingelfinger, 2008). In 2009, a World Health Organization expert meeting report recommended limits of 1.0 mg kg 1 and 2.5 mg kg 1 for melamine in powdered infant formula and in other foods, respectively, in order to provide a sufficient margin of safety for dietary exposure * Corresponding author. Departamento de Química Analítica, Instituto de Quí- mica, Universidade Federal Fluminense, Outeiro de S~ ao Jo~ ao Batista, s/n, 24020e141, Valonguinho, Centro, Niter oi, RJ, Brazil. Tel: þ55 (21)2629 2355; fax þ55 (21) 2629 2143. E-mail address: annibal@vm.uff.br (A.D. Pereira Netto). Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont http://dx.doi.org/10.1016/j.foodcont.2014.12.001 0956-7135/© 2014 Published by Elsevier Ltd. Food Control 51 (2015) 402e407