A Simple HPLC-DAD Method for the Analysis of Melamine in Protein Supplements: Validation Using the Accuracy Profiles

The study presents a fully validated simple high-performance liquid chromatography method with diode array detection (HPLCDAD), able to accurately determine the melamine, fraudulently added, in protein supplements, commonly used fromhealthy adults to enhance exercise or sport performance. The validation strategy was intentionally oriented towards routine use and the reliability of themethod rather than extreme performance. For this reason, validation by accuracy profile, including estimation of uncertainty, was chosen. This procedure, based on the concept of total error (bias + standard deviation), clearly showed that thismethodwas able to determine melamine over the range of 0.05–3.0mg Kg−1, selected by taking into account the maximum residue levels (MRLs) proposed by European legislation to distinguish between the unavoidable background presence of melamine and unacceptable adulteration.The accuracy profile procedure established that at least 95% of the future results obtained with the proposed method would be within the ±15% acceptance limits of the validated HPLC-DAD method over the whole defined concentration range. 1. Introduction Melamine (2,4,6-triamino-1,3,5-triazine) is commonly used as a rawmaterial or additive in themanufacture ofmelamineformaldehyde plastics or can coatings for food contact materials. It canbe foundat low levels as a contaminant in a variety of foods due to the leaching from food contact materials [1– 4]. For its nephrotoxicity [5–10] this chemical should not be intentionally added into foods or edible materials, in any circumstances. On the contrary, it is fraudulently added to food and/or feed ingredients to boost their total protein content and increase their market value. Such adulteration exploits a limitation of themost common nonspecific protein tests such as Kjeldahl method, which use total nitrogen as an indicator of protein content (melamine contains 66.6% of nitrogen by weight). In recent years the illegal addition of melamine to animal feedstuffs and infant milk formula caused illness and deaths of human infants and pet animals, who had taken the contaminated foods, primarily as a result of the accumulation of melamine-uric acid crystals in the urinary tract [8, 11– 15]. That is why various international health organizations have established regulations on melamine content over a wide variety of foods in order to safeguard public health [9, 16, 17]. In order to harmonize maximum residue levels (MRLs), set in many countries all over the world, European Food Safety Authority has proposed a legal maximumresidue level (MRL) of 1.0mg/Kg for infant foods and 2.5mg/Kg for other foods to distinguish between the unavoidable background presence of melamine (fromfood contact materials, pesticide use, etc.) and unacceptable adulteration [18]. Protein supplements are largely used by athletes and physically active individuals to enhance nitrogen retention, to increase muscle mass, to prevent protein catabolism during prolonged exercise, and to promote muscle glycogen resynthesis following exercise. Therefore it is likely that the adulteration of this kind of supplements with melamine increases their apparent protein content. In the