Studies On Confirmation Analysis Of Five Tetracyclines And Its Three Metabolites In Chicken Muscle Via HPLC-MS/MS

A high performance chromatography-tandem mass spectrometry(HPLC-MS/MS)method for simultaneous determination of the residues of Tetracyclines,Chlortetracycline,Oxytetracycline,Doxycycline,Tigecycline,4-epitetracycline,4-epichlortetracycline and 4-epioxytetracycline in chicken muscle had been established and validated,using Haiyang yellow chicken as experimental material in this study.This study provides scientific basis for the establishment of multi-residue testing standards for TC,CTC,OTC,DOXY,TGC,ETC,ECTC and EOTC in chicken muscle.The main results of this study are shown as follows:1.A pre-treatment extraction method of the residues of five tetracyclines and its three metabolites in chicken muscle was established.The extraction method is high extraction efficiency and the recovery rate is above 88.95%.Therefore,this pre-treatment extraction method can be successfully applied to the detection of multiple residues of tetracyclines and its metabolites in chicken muscle.2.A HPLC-MS/MS method was established for simultaneous extraction and confirmation of residues of TC,CTC,OTC,DOXY,TGC,ETC,ECTC and EOTC in chicken muscle.The chromatography was conduct on an Waters SunFireTMC8(5 ?m,50 mm×2.1 mm i.d.)at 28?,The mobile phase used in the gradient of elution was composed of methanol(A)and water containing 10 mmol/L of trifluoroacetate(B)at 0.3 mL/min;Mass spectrometry conditions using triple quadrupole tandem mass spectrometry,The mass spectrometer was equipped with an ESI source operating in positive ion mode,Multi-reaction monitoring(MRM).At the fortified levels of 0.15-200 ?g/kg for TC,0.13-200 ?g/kg for CTC,0.16-200 ?g/kg for OTC,0.12-200 ?g/kg for DOXY,0.19-200 ?g/kg for TGC,0.17-200 ?g/kg for ETC,0.16-200 ?g/kg for ECTC and 0.18-200 ?g/kg for EOTC in blank chicken muscle matrix,the peak area ratio of qutantificational ion showed a linear correlation with the concentration.The coefficients of determination(R2)of eight analytes in chicken muscle are higher than 0.9999,0.9997,0.9998,0.9997,0.9997,0.9998,0.9998 and 0.9999,respectively.At spiked levels of LOQ,0.5,1.0,2.0 fold of max residue limit(MRL),recoveries of TC,CTC,OTC,DOXY,TGC,ETC,ECTC and EOTC simultaneously were 89.61%-96.20%,89.43%-96.76%,90.97%-97.61%,91.37%-95.35%,88.95%-93.64%,90.37%-94.36%,93.26%-97.50%and 89.53%-94.37%;with the relative standard deviations(RSD)of 3.96%,4.15%,4.42%,3.39%,3.19%,3.28%,3.10%and 3.51%,respectively;intra-day RSD were less than 3.79%,4.92%,4.01%,5.02%,3.82%,4.13%,4.01%and 4.52%,respectively;Inter-day RSD were no higher than 5.96%,6.92%,5.87%,5.91%,5.76%,6.17%,6.51%and 6.94%,respectively;Limits of detection(LODs)and the highest values were 0.09,0.08,0.06,0.08,0.07,0.08,0.07 and 0.06 ?g/kg,respectively;Limits of quantitation(LOQs)and the highest values were 0.15,0.16,0.13,0.12,0.19,0.17,0.18 and 0.16 ?g/kg,respectively;The CC? and CC? were all near the MRLs in chicken muscle.The established HPLC-MS/MS method was successfully applied in practice of determination of five tetracyclines(TC,CTC,OTC,DOXY and TGC)and its three metabolites(ETC,ECTC and EOTC)residue in chicken muscle.