Determination Of Nitroimidazole Residues In Swine Tissues By A HPLC Method And A GC-MS/MS Method

5-Nitroimidazoles had been widely used in veterinary medicine for treatment of bacterial and protozoal infections in poultry (histomoniasis in turkey et al.) and swine (swine dysentery). Any residues of these compounds were not permitted in food-producing animals by many countries because their mutagenic and potential carcinogenic properties. Now there have no methods for the quantification of these compounds in our country (mainland). The aim of the study was to establish two methods for the determination and confirmation of hydroxydimetridazole(HMMNI), ronidazole(RNZ), metronidazole(MNZ), tinidazole(TNZ), dimetridazole(DMZ), ornidazole(ONZ) and secnidazole(SNZ) in swine muscle, fat, liver and kidney.In the HPLC method, the nitroimidazole residues were extracted with dichloromethane, followed by reextracted with hydrochloric acid. The acid extract was made basic with K₂HPO₄, followed by extracted with dichloromethane. The final solution was carefully evaporated just to dryness with a vacuum evaporator (water bath at 40℃). The remaining residues were mixed with 0.5mL mobile phase. Detection wavelength was set at 320nm and the injection valume was 20μL. The mobile phase was methanol and water (16:84 v/v). The limits of detection and quantitation for standard solution of HMMNI, RNZ and MNZ were 0.005μg/mL and TNZ, DMZ, ONZ are 0.01μg/mL. The calibration curve was in linear with a correlation coefficient (r) of over 0.996 in range of 0.005 ².0μg/mL. The limits of detection and quantitation were 0.5μg/kg for HMMNI, RNZ, MNZ and 1.0μg/kg for TNZ, DMZ, ONZ in muscle and fat, 1.0μg/kg for HMMNI, RNZ, MNZ, TNZ, DMZ and ONZ in liver and kidney. The control samples were all spiked with nitroimidazoles at levels of 0.5⁴μg/kg. The mean recoveries of nitroimidazoles were 60% 83 %, the inter-day relative standard deviation was less than 15%.In the GC-MS/MS method, the nitroimidazole residues were extracted with dichloromethane, followed by reextracted with hydrochloric acid. The acid extract was made basic with K₂HPO₄, followed by extracted with dichloromethane. The final solution was carefully evaporated just to dryness with a vacuum evaporator (water bath at 40 ℃). The remaining residues were dissoluted in 3mL ethyl acetate. The solution wastransferred into a derivation tube, evaporated to dryness under a weak nitrogen stream and derivatised with a solution of 50^1 of BSA-50fil of i-octane for 60 min at 50°C to produce trimethylsilyl-derivatives(TMS-derivatives). The resulting mixture was directly injected into the GC-EI-MS/MS system. VF-5 GC-MS column was used. A Varian 2200 mass spectrometer coupled with a Varian 3800 GC system. The parent ions of DMZ, RNZ, SNZ, MNZ and ONZ were 141, 214, 181, 182 and 276 respectively. The intensity of the characteristic ions at m/z of 95, 111, 112, 141 and 142 for DMZ; 153, 167, 168 and 215 for RNZ; 112, 140 and 182 for SNZ; 112, 140, 167 and 182 for MNZ; 215, 230 and 276 for ONZ were monitored and several of them were choiced for quantitation. The calibration curve was in linear with a correlation coefficient (r) of over 0.998 in range of 0.005 1.6u.g/mL. The limits of detection and quantitation were 0.2p.g/kg for RNZ, 0.5(ig/kg for SNZ, 1 .Op.g/kg for DMZ, MNZ and ONZ. Recoveries of five nitroimidazoles in tissues spiked at 0.2-^ug/kg are 69 % 82 %, the inter-day relative standard deviation was less than 15%.The two methods were simple, rapid and no extensive sample clean-up was necessary. The HPLC method was suitable for routine analysis of a large number of samples. Because the specificity and sensitivity of the GC- MS/MS method, and it was the first time found in China(mainland). It's well adapted for confirmation of banned substances.