Studies On Methodology For The Multiresidue Analysis Of Amoxicillin, Its Major Metabolites, Ampicillin And Tissue Depletion Of Amoxicillin And Its Major Metabolites In Chicken

A HPLC-MS/MS method and a UPLC-MS/MS method for simultaneous determination of amoxicillin (AMO), its major metabolites (amoxicilloic acid (AMA), amoxicillin diketopiperazine-2’,5’-dione (DIKETO) and ampicillin (AMP) in chicken tissues had been developed and validated, using Jinghai yellow chicken as experimental material in this study. At the same time, the study on elimination of residues of AMO, AMA and DIKETO has been conducted in chicken tissues, after the chicken orally administered successively AMO capsules of30.0mg/kg,60.0mg/kg of body weight twice every day for7days, respectively. The study not only provides basis for the proper use of AMO, but also estabilishing withdrawal time and standards of determination of AMO, AMA, DIKETO and AMP in foodstuffs of animal orgin and developing pollution-free livestock and poultry products. The main results were as follow:1. A HPLC-MS/MS method was developed for simultaneous extraction and conformation of AMO, AMA, DIKETO and AMP in chicken tissues. At the fortified levels of every specified concentration range for AMO, AMA, DIKETO and AMP in the three different tissues, the peak area ratio of qutantificational ions of the target analytes to qutantificational ion of internal standard (PV) showed a linear correlation of the concentration ratio of the analytes to PV. The coefficients of determination (R2) of four analytes are all higher than0.9998,0.9997,0.9989and0.9968in the different tissues. At the fortified levels of LOQs for AMO, AMA, DIKETO and AMP in specified tissues, the recovery values of four analytes in muscle, liver and kidney are higher than75.20%,79.62%,79.85%and79.65%, respectively. The relative standard deviations (RSD) are all less than16.35%,14.16%,16.08%and14.88%. At the fortified levels of25μg/kg,50μg/kg and100μg/kg for both AMO, AMA, DIKETO and AMP, the average recovery rates in the three tissues range90.83%to106.32%for AMO,83.09%to103.56%forAMA,93.52%to104.50%for DIKETO and86.09%to107.62%for AMP, respectively. In other words, the accuracy ranges of the method in three biological matrixes are-9.17%～+6.32%for AMO,-16.91%～+3.56%forAMA,-6.48%～+4.50%for DIKETO and-13.91%～+7.62%for AMP, respectively. The relative standard deviations (RSD) are all less than13.72%,14.77%,14.62%and12.03%, respectively. The corresponding intra-day and inter-day variation (RSD) were found to be less than11.09%and12.66%for AMO,12.07%and15.39%for AMA,13.73%and15.33%for DIKETO,14.16%and15.00%for AMP,respectively. Under the condition of HPLC-MS/MS detection technology, the maximum of limits of detection (LODs) in three tissues were1.20,2.20,0.46and0.30μg/kg for the analytes (S/N=3); the maximum of limits of quantitation (LOQs) in three tissues were4.50,8.50,1.38and0.90μg/kg for the analytes (S/N=10).2. A UPLC-MS/MS method was developed for simultaneous conformation and quantitation of AMO, AMA, DIKETO and AMP in chicken tissues. At the fortified levels of every specified concentration range for AMO, AMA, DIKETO and AMP in the three different tissues, the peak area ratio of qutantificational ions of the target analytes to qutantificational ion of internal standard (PV) showed a linear correlation of the concentration ratio of the analytes to PV. The coefficients of determination (R2) of four analytes are all higher than0.9994,0.9999,0.9998and0.9989in the different tissues. At the fortified levels of LOQs for AMO, AMA, DIKETO and AMP in specified tissues, the recovery values of four analytes in muscle, liver and kidney are higher than72.05%,74.85%,75.43%and79.18%, respectively. The relative standard deviations (RSD) are all less than15.73%,16.39%,14.30%and13.91%. At the fortified levels of25μg/kg,50μg/kg and100μg/kg for both AMO, AMA, DIKETO and AMP, the average recovery rates in the three tissues range84.08%to102.11%for AMO,88.85%to104.08%forAMA,86.73%to108.13%for DIKETO and84.31%to102.69%for AMP, respectively. In other words, the accuracy ranges of the method in three biological matrixes are-15.92%～+2.11%for AMO,-11.15%～+4.08%for AMA,-13.27%～+8.13%for DIKETO and-15.69%～+2.69%for AMP, respectively. The relative standard deviations (RSD) are all less than13.03%,12.33%,12.67%and12.56%, respectively. The corresponding intra-day and inter-day variation (RSD) were found to be less than11.77%and10.95%for AMO,14.87%and15.16%for AMA,11.34%and11.27%for DIKETO,10.59%and12.18%for AMP, respectively. Under the condition of UPLC-MS/MS detection technology, the maximum of limits of detection (LODs) in three tissues were0.68,1.36,0.07and0.05μg/kg for the analytes (S/N=3); the maximum of limits of quantitation (LOQs) in three tissues were2.72,5.44,0.23and0.25μg/kg for the analytes (S/N=10).3. A study on HPLC-MS/MS method and elimination of residues of AMO and its major metabolites (AMA and DIKETO) was conducted in muscle, liver and kidney of Jinghai yellow chicken using HPLC-MS/MS, after the chicken orally administered successively AMO capsules of30.0mg/kg,60.0mg/kg of body weight twice every day for7days, respectively. The maximum residues of AMO, AMA and DIKETO were both detected at a withdrawal time of4h in the three tissues, and the phenomenon of every analyte distribution:kidney>liver>muscle; During the period of4h of the withdrawal time to1d of the withdrawal, the concentration of AMO, AMA and DIKETO in all tissues depleted rapidly, and the residue elimination rate of all the drugs in this three tissues is for kidney>liver>muscle; In the early withdrawal, AMA metabolite residues detected in all tissues was significantly higher than that of AMO prototype and DIKETO metabolite, and AMO prototype which has kept broadly in step with DIKETO metabolite residued in tissues; After a withdrawal time of Id, the analytes residue elimination rates significantly decreased in this three tissues; Residues of AMO, AMA and DIKETO in muscle, liver and kidney were both positively correlated with AMO orally administered doses during the early of withdrawal, but this phenomenon is not obvious during the later of withdrawal.4. With the reference of the criterions of the maximum residue limits (MRLs) of China and European Medicines Agency (EMEA) in the edible animal tissues about AMO. Linear regression analysis of the logarithmic transformed data (sum concentration of AMO, AMA and DIKETO) can be censidered for the calculation of the withdrawal periods using the WT1.4software. The calculation results show that when the same MRL of AMO should be used for the withdrawal time calculation, a withdrawal time of5.58,9.58days for chicken muscle,7.79,11.82days for liver,7.42,9.38days for kidney can be calculation for orally administered of30.0,60.0mg/kg of AMO of body weight twice every day for7days, respectively. Finally, we suggested that the withdrawal time should be8d,12d, respectively. To guarantee food safety, this research suggested identification and monitoring AMA and DIKETO as marker residue of AMO in China.