Determination of robenidine in eggs by liquid chromatography with UV spectrophotometric detection
Introduction
Anticoccidials are compounds used extensively both prophylactically and therapeutically to treat coccidiosis in poultry. Coccidiosis is caused by unicellular organisms belonging to the genus Eimeria in the class Sporozoa and is the most important parasitic disease of poultry [1]. Robenidine (1,3-bis[(p-chlorobenzylidene) amino]-guanidine monohydrochloride) is a common anticoccidial drug used in the poultry industry. The molecular structure of the compound is shown in Fig. 1. It is authorised for use in poultry and other species under the Additives Directive 70/524/EEC [2]. In the European Union, according to Directive 70/524/EEC, the use of robenidine is not authorised for laying hens. The widespread use of this anticoccidial drug may present a potential risk to the consumer if residues enter the food chain. A range of methods have been described for determination of robenidine in feed, including thin layer chromatography [3], a photometric method for feed and premixes [4], and a LC-UV method for determination of robenidine and its chemical precursors in feed [5]. A polarographic method for determination of robenidine in tissue, eggs, litter and soil [6], and methods for determination of robenidine and other anticoccidials by LC–dual mass spectrometry (MS/MS) in eggs [7], [8] have been described. There are no methods available for determination of robenidine in egg by LC with UV detection.
In this paper a fast, simple and reliable LC method is described for the detection of robenidine in eggs. The method was validated according to procedures described in Commission Decision 2002/657/EC [9].
Section snippets
Materials and reagents
Water (HiPerSolv grade), acetonitrile (HiPerSolv grade), n-hexane, triethylamine and ammonium dihydrogen phosphate (Analytical grade) were obtained from BDH (Poole, Dorset, UK). N,N-dimethylformamide (Analytical grade) was obtained from Riedel-de-Haen (Seezle, Hannover, Germany). Robenidine hydrochloride was obtained from QMX (Essex, UK). Ammonium phosphate buffer (0.05 M, pH 6.8) was prepared by dissolving 5.75 g of ammonium dihydrogen phosphate in 800 ml LC grade water then adding triethylamine
Preliminary experiments
An LC method, developed to analyse for robenidine in animal feed, was applied to the separation of robenidine on a C18 column with a mobile phase consisting of acetonitrile:water:potassium di-hydrogen phosphate:di-sodium hydrogen phosphate (65:25:5:5) [5]. The peak shape was poor, with excessive tailing, using this mobile phase. Investigation of various LC conditions, including various percentages of organic content in the mobile phase, pH, injection volume and mobile phase flow rate, showed
Conclusions
This paper reports the development and validation of a fast and simple LC method for the determination of robenidine in eggs. The method was validated according to the guidelines described in Commission Decision 2002/657/EC.
Acknowledgements
This work was funded by the Teagasc Walsh Fellowship programme. The authors thank staff at The National Food Centre, Teagasc, Dublin for their practical assistance.
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