Determination Of Metronidazole,Dimetridazole And Ipronidazole In Feeds By HPLC Method

Nitroimidazoles drugs, with 5-nitro imidazole ring structure, are widely used to prevent and treat the trichomoniasis of poultry, hemorrhagic diarrhea and anaerobic infections of swine, as well as serve as feed additivities to promote animals growth. Metronidazole(MNZ), dimetridazole (DMZ) and ipronidazole (IPZ) are the most popular nitroimidazoles used as additivites. However, it has been reported that nitroimidazoles have the potential effect in carcinogenicity, teratogenicity and mutagenicity and animal tissue residues cause a serious threat to food security so that the EU and American have prohibited the use of nitroimidazoles in feed additivives, and these drugs can not be detected in edible animal tissues. Similarly, MNZ, DMZ and their salt, ester as well as preparations are prohibited from being used in food animals for growth promotion in China. Thus, it is quite necessary that developing the nitroimidazoles testing method which is simple, convenient, quick and accurate, High-performance liquid chromatography (HPLC) is commonly used to determine nitroimidazoles in feeds. However, the conventional HPLC approach only ascertains one or two kinds of nitroimidazoles in feeds with lower sensitivity. This study aims at developing a simple and effective method with reproducibility for simultaneous determination of MNZ,DMZ and IPZ in feeds.1. Optimizing the chromatographic conditions and pretreatment methodsThe study has determined the sample pretreatment conditions and the chromatographic conditions. The feed samples pretreatment conditions are as follows Samples were extracted with ethyl acetate, and the extract was evaporated just to dryness with a pressure blowing concentrator (water bath at 35 ℃). The residue was dissolved in hexane and back extracted with phosphoric acid solution (0.1 mol/L). Then the obtained solution was purified using mixed-mode cation exchange (MCX) solid-phase extraction (SPE) column and filtered using a 0.22 μm filter. The HPLC conditions for determination of MNZ, DMZ and IPZ in feeds are as follows:The extracts separated with UV detector 320 nm pumping at a flow rate of 0.8 ml/min. The liquid chromatographic separation of the extracts was performed on a C18 bonded silica column (4.6 x 250 mm,5 μm) applying a gradient of elution with water (A) and methanol (B).2. Method Verification.The method built above was validated by evaluating its specificity, linearity, sensitivity, accuracy and precision. The results are as follows:The specificity of the method performed well. The linearity was satisfactory with a correlation coefficient of ≥0.9990 at concentrations ranging from 0.02 to 10 mg/kg. The limit of detection of MNZ, DMZ and IPZ was 0.01 mg/kg, and the quantitation limit was 0.02 mg/kg. The recoveries of the MNZ, DMZ and IPZ for feeds samples were in the range of 90.6%-107.7%. The intra-day precision (n=5) for nitroimidazoles in feeds spiked at 0.05, 0.5,5 mg/kg were in the range of 1.2%-12.4% and the inter-day precision for 3 days (n=5) was 2.8%-9.7%. This method was simplicity of operator with accurate results, thus could be used to simultaneously determine MNZ, DMZ and IPZ in feeds.