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

A high-performance liquid chromatographic (HPLC) method based on solid phas e extraction was developed for determination of metronidazole (MNZ) and its metabolite hydroxymetronidazole (MNZ-OH) in muscle and skin tissues of rai nbow trout. Tinidazole (TNZ) was used as internal standard. The compounds w ere extracted with acetonitrile and the extract was evaporated and redissol ved 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 analys ed by reverse phase gradient HPLC on a C18 column followed by ultraviolet d etection at 325 nm. The limit of detection was 2.8 mu g/kg for both compoun ds in muscle. The estimated limits of detection in skin tissue were 3 mu g/ kg for MNZ and 5 mu g/kg for MNZ-OH. The mean recoveries of MNZ in muscle c alculated without use of internal standard were 93% and 81% at levels of 10 mu g/kg and 25-100 mu g/kg respectively. The mean recovery of MNZ-OH in mu scle was 79% at a level of 10-100 mu g/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 10-100 mu g/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 administratio n period but not 3 weeks later.