Development Of A High-performance Liquid Chromatographic Method For Detection Of Metronidazole Residues In Fish Edible Tissues, Eggs And Milk

Metronidazole is a nitroimidazole compound active in treatment of anaerobic protozoan and bacterial infection. It is considered to be carcinogenicity and maximum residue limit in any animal edible tissue is zero. This study is aimed at developing a reverse phase high performance liquid chromatographic method (RP-HPLC) to detect metronidazole residues in fish tissues, eggs and milk. The compound is extracted with acetonitrile in fish tissues,and with ethyl acetate in eggs and milk. Then the extract is evaporated and redissolved in a mixture of hexane and ethyl acetate(2+l). The mixture is cleaned up by solid phase extraction on a silica cartridge. The eluate is evaporated to dryness with nitrogen followed by dissolution with HPLC eluent. The final extract is analysed by RP-HPLC with ultraviolet detection at 325 nm. The liquid chromatographic seperation is made on C18 column applying a gradient with an acetate buffer with pH4.3 and acetronitrile(88+12). The limits of detection and quantitation for standard solution of metronidazole are 0.01ug/mL. The calibaration curve is linear with an correlation coefficient (R2)of lin range of 0.01 ~ 1.0ug/mL and 0.1 ~ 100.0,wg/mL. The inter-day reproducibilities of standard solution of metronidazole at three levels(0.01ug/mL, 1.0ug/mL and 100.0ug/mL) are respectively 1.5%, 2.7%, 5.8% and intra-day reproducibilities respectively 2.5%% 7.1%% 9.4%. The limits of detection and quantitation in fish tissues , eggs and milk are lug/kg. The control samples are all fortified with MTZ at levels of 1ug/kg, 2ug/kg, and 4ug/kg. The mean recoveries of metronidazole in milk respectively are respectively 65.7% +7.4%, 67.7% +6.4%, 67.6% +4.9% in fish muscle; respectively 65.4% +5.0%, 66.5% +5.0%, 68.2% +4.3% in fish skin; 75.4% +8.7%, 79.0% +7.0%, 81.9% +5.2%; respectively 68.8% +6.7%, 72.4%+6.8%, 78.3% +6.4% in eggs. The inter-day relative deviation is less than 10% and the intra-day relative deviation is less than 15%. The method was applied to laying hens given feed containting 0.025% metronidazole for 7 days. Residues of metronidazole and its metabolite were detected in eggs shortly after the administration period until 14 days later. The concentration of the compound in eggs was caculated with external standard. Withdrawal time of MTZ in eggs is 15 days by log-transformed concentration of MTZ in eggs vs time. The results shows that this method is accurate and feasible enough to determination the residues of metronidazole with simple pre-treatment and can be applied on institutes of veterinary drug control, food quarantine departments, trade ports and other institutes.