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HPLC Analysis of Melamine and Related Substances in Fertilisers
by Azusa Morita, Shimadzu Corporation, Japan Gesa J. Schad, Shimadzu Europa GmbH
Shirai Yuji, Food and Agricultural Materials Inspection Center, Japan
Calcium cyanamide is a multipurpose nitrogen fertiliser, which is also effective as a pesticide and soil amendment. It plays an important role in crop cultivation by ensuring that plants are supplied with nitrogen. Recently, high levels of melamine were discovered as a by-product in some calcium cyanamide hydrate products, pelletised by added water. Due to the risk of agricultural products absorbing melamine from soil, it has been identified as a potential public health risk.
Health issues associated with melamine have been in the public eye since the Chinese milk scandal in 2008. A widely utilised precursor for many applications on the one hand, harmful contamination for feedstock and milk on the other, its use in fertiliser can also facilitate the introduction of melamine into the food chain. Long-term exposure to melamine and its related substance, cyanuric acid, through ingestion can result in bladder or kidney stones which ultimately can lead to bladder cancer [1, 2], concentration of this compound in food and feed needs to be controlled carefully.
The Food Safety and Consumer Affairs Bureau in the Ministry of Agriculture, Forestry and Fisheries in Japan issued a notice specifying a 0.4% provisional maximum allowable concentration of melamine in calcium cyanamide [3].
In this work, a simple and robust method for sample pretreatment and analysis of melamine and related substances, namely ammeline, ammelide and cyanuric acid in fertiliser, carried out using HPLC with UV detection is demonstrated. Five products were investigated, commercially available in Japan, namely Nitrolime 1, Nitrolime 2, synthetic fertilisers (x 2) and ammonium sulphate.
Materials and Methods
Melamine synthesis can result in a number of by-products by replacement of the amino group ‘-NH2’ in the R1-R3 positions with a hydroxy group ‘-OH’. The structures of melamine and its related substances are shown in Figure 1.
Figure 1: Chemical structures of melamine and its related substances.
Simultaneous analysis of the compounds of interest was performed using a Shimadzu Prominence™ HPLC system consisting of a DGU-20A degassing unit, an LC-20AD pump, a SIL-20ACHT auto-sampler, a CTO-20AC column oven and an SPD-20A UV-visible detector. Chromatographic separations were performed using a TSKgel® Amide-80 column (250 mm L. x 4.6
mm I.D., 5 µm) at a temperature of 40°C. Analytical conditions are further specified in Table 1.
Sample preparation
Fertiliser samples to be tested were prepared in accordance with the testing methods for fertilisers [4]. Five hundred
Table 1: Analytical conditions of the analysis of melamine and related substances.
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