Multi-residue Determination Of 11 Quinolones In Animal Tissues By High Performance Liquid Chromatography

Quinolones (QNs) antibiotics are widely used for veterinary applications andaquaculture. As a consequence the increased use of quinolone can promote the resistanceof bacteria. To protect the consumers health., Maximum Residue Limits(MRL) have beenestablish in animal foods by the Ministry of Agriculture. The aim of the study was todevelope the method for simultaneous determination of norfloxacin (NOR), ofloxacin(OFL),ciprofloxacin(CIPRO),pefloxacin(PEF),lomefloxacin(LOME),danofloxacin(DANO), sarafloxacin(SARA), enrofloxacin (ENRO),difloxacin (DIF),oxolinic acid (OXA) andflumequine (FLU) residues in bovine muscle, swine muscle, liver and kidney by highperformance liquid chromatography(HPLC), and settle a basis for establishing the nationalstandard of detecting these durgs.Sample Pretreatment Methods: The samples were extracted by 10% trichloroaceticacid (in water)/acetonitrite (9:1, v/v) and (8:2, v/v),then diluted to acetonitrile contentunder 10% with H20,cleaned by Strata X reverse phase solid-phase extraction (SPE)cartridges. The procedure of clean-up: the cartridges were conditioned with 3 ml ofmethanol and 3 ml distilled water, and loaded, and then rinsed with 3 ml of 10% methanolin water, finally eluted with 3 ml of methanol. The eluates were evaporated to dryness anddissolved the residue with mobile phase.Analytical Condition of HPLC: The quinolones were separated on a reverse phaseC18 column (Hypersil BDS-C18) with mobile phase B (0.05mol/Lcitric acid/0.1 mol/Lammonium acetate/ triethylamine pH4.3-4.5—acetonitrile)linear gradient elution anddetected with fluorescence detection by means of a wavelength program. The otherchromatographic conditions were: the flow-rate of mobile phase was 2.0 ml/min, and thecolumn temperature was maintained at 50℃, and the stop time was 27 min, and intervalbetween runs was 3min; the gradient program, combining ACN and solution B, wasused: 8%-24% ACN (0-21 min), and 24%-40% ACN(21-27.0 min),then8%ACN(27.01 min) ; the fluorescence excitation/emission wavelengths were 312/366 nm forthe analysis of OXA and FLU, and 278/465 nm for the other 9 QNs. The results of the method for simultaneous determination of 11 QNs in bovine muscle.swine muscle, liver and kidney are as follows:The recoveries for bovine muscle fortified with 11 QNs at three levels (1/2MRL.MRL.2MRL) were 50.47%～101.03％with acceptable intra-day C.V.≤8.54％and inter-dayC.V.≤11.30％.The total average recoveries(%) and inter-day C.V.(％)of each QNs were58.24,9.94 for NOR;83.33,6.19 for OFL; 73.85.7.23 for CIPRO;84.63,4.41 for PEF: 82.55,4.53 for LOME;87.50,3.98 for DANO,91.23,5.20 for ENRO;85.15,3.57 for SARA; 84.56,4.59 for DIF; 80.15, 5.16 for OXA;82.24,4.20 for FLU.The recoveries for swine muscle fortified with 11 QNs at three levels (1/2MRL, MRL,2MRL) were 51.96%～92.70%with acceptable intra-day C.V.≤9.69% and inter-day C.V.≤13.46%. The total average recoveries(%) and inter-day C.V.(%)of each QNs were 68.76,11.50 for NOR; 89.46, 5.75 for OFL; 75.14,7.08 for CIPRO; 84.95,5.14 for PEF; 85.07,4.11for LOME; 81.90,4.92 for DANO,81.35,5.99 for ENRO;73.58,7.01 for SARA;77.65, 8.57for DIF; 71.50,6.76 for OXA;75.82,3.59 for FLU.The recoveries for swine liver fortified with 11 QNs at three levels (1/2MRL, MRL,2MRL) were 61.65%～98.49% with acceptable intra-day C.V.≤8.47 % and inter-day C.V.≤10.21%. The total average recoveries(%) and inter-day C.V.(%)of each QNs were 66.00,8.59 for NOR;89.45,4.82 for OFL; 68.58,6.33 for CIPRO;76.63,5.03 for PEF; 86.32, 6.98for LOME;81.41,8.39 for DANO;82.16,7.26 for ENRO;76.97, 7.35for SARA; 77.75, 5.02for DIF;72.34,6.62 for OXA;74.14,5.30 for FLU.The recoveries for swine kidney fortified with 11 QNs at three levels (1/2MRL, MRL,2MRL) were 55.50%～98.34%with acceptable intra-day C.V.≤8.02 % except FLU C.V.≤12.09% and inter-day C.V.≤12.16%. The total average recoveries(%) and inter-day C.V.(%) of each QNs were 67.11,7.13 for NOR;85.95,8.62for OFL;73.37,11.18 for CIPR;84.95 5, 8.98 for PEF;84.60,11.31 for LOME;88.12,9.09for DANO;85.68,8.84 forENRO;83.37,10.67 for SARA; 81.68, 9.72 for DIF;72.43,5.54 for OXA;82.8,7.45 for FLU.Limit of detection(LOD) and Limit of quantification (LOQ): 0.2～6.5μg/kg and0.7～21.6μg/kg in bovine muscle; 0.2～6.0μg/kg and 0.8～20.0μg/kg in swine muscle;0.3～10.0μg/kg and 1.2～33.3μg/kg in swine liver, 0.3～8.2μg/kg and 0.9～27.2μg/kgin swine kidney.It was the first time to develop the method for simultaneous determination of 11 QNsresidues in animal tissues by HPLC in china. The results shows that this method is accurate and feasible enough to determination the residues of quinolones and can be applied oninstitutes of veterinary drug residual control. food quarantine departments, trade ports andother institutes.