Simultaneous determination of triclabendazole and its sulphoxide and sulphone metabolites in bovine milk by high-performance liquid chromatography
Introduction
Triclabendazole is a benzimidazole anthelmintic widely used in the control of the liver fluke, Fasciola hepatica, in sheep and cattle. After oral administration, it has been found that triclabendazole is oxidized to sulphoxide and sulphone metabolites in animals, similarly to other benzimidazole sulphide compounds [1], [2], [3] (Fig. 1.)
FAO/WHO had reported the recommended maximum residue limits (MRLs) for triclabendazole in animal foods [4]. The MRLs of triclabendazole are scheduled to be adopted after June of 2000 in Japan. In order to monitor the residual triclabendazole and its metabolites in commercial milk samples, a simple, rapid and sensitive method is needed.
Methods have been published for the determination of triclabendazole and its metabolites in biological samples using HPLC with UV or fluorescence detection in pharmacokinetic studies [1], [2], [5], [6], [7], [8].
A method has been published for the determination of eight benzimidazole anthelmintics including triclabendazole in meat samples [9]. But, the metabolites of triclabendazole could not be simultaneously studied by this method. At present no method is available for the simultaneous determination of triclabendazole and its metabolites in milk.
The purpose of this study was to develop a simple method for the simultaneous determination of triclabendazole and its metabolites in milk.
Section snippets
Materials and reagents
Triclabendazole and its metabolites (sulphoxide and sulphone) were kindly supplied by Ciba-Geigy (Munchwilen, Switzerland).
Acetonitrile and n-hexane were of HPLC grade (Kanto, Tokyo, Japan). Sodium sulfate anhydrous, potassium phosphate monobasic, sodium hydrogencarbonate and ammonium acetate were of analytical-reagent grade (Wako, Osaka, Japan). Distilled water was of HPLC grade (Kanto).
Solid phase extraction C18 cartridge was of Bond Elut LRC, 500 mg (Varian, Harbor City, CA, USA). The
Chromatographic conditions
To achieve an efficient separation of the triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone, we investigated the retention factor (k) of these compounds in the mobile phase by changing the concentration (40–70%) of acetonitrile. The relationship between the retention factor (k) and concentration of acetonitrile in the mobile phase is shown in Fig. 2.
Because the k value of triclabendazole was remarkably increased when there was <50% acetonitrile in the mobile phase, it was
Conclusions
We described a rapid and sensitive HPLC method that allowed for the simultaneous determination of triclabendazole and its metabolites in milk using a simple sample preparation procedure and isocratic mobile phase with UV detection. The analysis time was ∼15 min. The mean recoveries were > 89% with RSD values within 3%. The detection limits were 0.004–0.006 μg/g. The proposed method may be applicable to the monitoring of triclabendazole and its metabolites (sulphoxide and sulphone) in milk.
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