Determination of metronidazole and hydroxymetronidazole in trout by a high-performance liquid chromatographic method

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Abstract
A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for determination of metronidazole (MNZ) and its metabolite hydroxymetronidazole (MNZ-OH) in muscle and skin tissues of rainbow trout. Tinidazole (TNZ) was used as internal standard. The compounds were extracted with acetonitrile and the extract was evaporated and redissolved in a mixture of ethyl acetate and hexane (1:2). The extract was cleaned up by solid phase extraction on a silica cartridge. The extract was analysed by reverse phase gradient HPLC on a C18 column followed by ultraviolet detection at 325nm. The limit of detection was 2.8 μg/kg for both compounds in muscle. The estimated limits of detection in skin tissue were 3 μg/kg for MNZ and 5 μg/kg for MNZ-OH. The mean recoveries of MNZ in muscle calculated without use of internal standard were 93% and 81% at levels of 10 μg/kg and 25-100 μg/kg respectively. The mean recovery of MNZ-OH in muscle was 79% at a level of 10–100 μg/kg. The mean relative repeatability standard deviations on spiked muscle tissue were 3.3% for MNZ and 3.2% for MNZ-OH at a level of 107–100 μg/kg. The method was applied to trout given feed containing MNZ in an aquaculture pilot plant. Residues of MNZ and MNZ-OH were detected in muscle and skin tissues shortly after the administration period but not 3 weeks later.