| A method of high perfonnance liquid chromatography (HPLC) with fluorescence detection (FLD) has been developed and validated for simultaneous determination of amoxicillin (AMO) and ampicillin (AMP) in eggs of laying hens, when Jinghai yellow chicken laying hens as experimental material in this study. At the same time, the study on elimination of residues of AMO and AMP has been conducted in the egg white, egg yolks and whole eggs. After the laying hens orally administered successively Amo solution of25mg·kg-1and Amp solution60mg·kg-1of body weight once every day for5days, respectively. The results indicated that:1.The method of sample pre-treatment and HPLC-FLD for determining AMO has been established in egg white、egg yolk and whole egg. The recovery for AMO was77.43%～89.67%in egg white, the intra-day relative standard deviation (RSD) and inter-day relative standard deviation (RSD) were less than9.63%and14.22%, respectively; The recovery for AMO was79.37%～88.79%in egg yolk, the intra-day RSD and inter-day RSD were less than9.71%and12.88%, respectively; The recovery for AMO was80.47%～89.53%in whole egg, the intra-day RSD and inter-day RSD were less than9.61%and14.80%, respectively. The linear range was5-800ng·mL-1of AMO (r=0.9997,n=8). The limit of detection and (LOD) and limit of quantitation (LOQ) for AMO were1.25and3.9ng·g-1, respectively. This method meets the demand of residues determination of AMO in eggs.2.The method of sample pre-treatment and HPLC-FLD for determining AMP has been established in egg white、egg yolk and whole egg. The recovery for AMP was79.12%-88.52%in egg white, the intra-day RSD and RSD were less than9.44%and13.34%, respectively; The recovery for AMP was78.64%～83.60%in egg yolk, the intra-day RSD and inter-day RSD were less than8.94%and12.36%, respectively; The recovery for AMP was78.30%～85.11%in whole egg. the intra-day RSD and inter-day RSD were less than9.64%and12.48%, respectively. The linear range was5～800ng·mL-1of AMP (r=0.9994,n=7). The LOD and LOQ for AMP were0.4and1.5ng·g-1, respectively. This method meets the demand of residues determination of AMO in eggs.3.The method of sample pre-treatment and HPLC-FLD for simultaneous detennination of AMO and AMP has been established in egg white、egg yolk and whole egg. The recoveries for AMO and AMP were80.53%～89.84%and80.08%～87.63%in egg white, respectively; The recoveries for AMO and AMP were78.24%～87.42%and77.79%～80.91%in egg yolk, respectively; The recoveries for AMO and AMP were78.40%～88.72%and77.60%-82.01%in whole egg respectively. The LOD for AMO and AMP were1.2and0.4ng·g-1, respectively; The LOQ for AMO and AMP were3.9and1.5ng·g-1, respectively. This method meets the demand of residues simultaneous determination of AMO and AMP in eggs.4. The study on elimination of residues of AMO has been conducted in the egg white, egg yolks and whole eggs. After the laying hens orally administered successively AMO solution of25mg·kg-1of body weight once every day for5days. The results indicated that:During medication period, residues of AMO in egg white, egg yolk and whole egg have a rise trend, the maximum residue concentration of AMO was detected at the1st day after withdrawal time in egg white and whole egg, but was detected2nd day after withdrawal time in egg yolk, they were13.1073ng·g-1、13.7757ng·g-1and16.0672ng·g-1respectively. And the content of AMO in egg yolk was more than the content of AMO in egg white. During withdrawal time, residues of AMO in egg white, egg yolk and whole egg have a degression trend, residues of AMO in egg white was lower than the LOD of AMO (1.2ng·g-1) at6th withdrawal day, but can still be detected in egg yolk and whole egg; residues of AMO in whole egg was lower than the LOD of AMO (1.2ng·g-1) at10th withdrawal day; residues of AMO in egg yolk was lower than the LOD of AMO (1.2ng·g-1) at12th withdrawal day. The residues of AMO in egg yolk eliminated slower than in egg white and long elimination time in egg yolk.5. The study on elimination of residues of AMP has been conducted in the egg white, egg yolks and whole eggs. After the laying hens orally administered successively AMP solution of60mg·kg-1of body weight once every day for5days. The results indicated that: During medication period, residues of AMP in egg white, egg yolk and whole egg have a rise trend, the maximum residue concentration of AMP was detected at the1st day after withdrawal time in egg white and whole egg, and was detected2nd day after withdrawal time in egg yolk, they were12.4136ng·g-1、12.2861 ng·g-1and17.2553ng·g-1respectively. And the content of AMP in egg yolk was more than the content of AMP in egg white. During withdrawal time, residues of AMP in egg white, egg yolk and whole egg have a degression trend, residues of AMP in egg white and whole egg were lower than the LOD of AMP (0.4ng·g-1) at9th withdrawal day, but can still be detected in egg yolk; residues of AMP in egg yolk was lower than the LOD of AMP (0.4ng·g-1) at10th withdrawal day. The residues of AMP in egg yolk eliminated slower than in egg white and long elimination time in egg yolk.6. With the reference of the criterions of the maximum residue limits (MRLs) of Japan in eegs about AMO and AMP, this research suggested when the laying hens orally administered successively AMO solution of25mg·kg-1of body weight once every day for5days, the withdrawal time should be5-day; when the laying hens orally administered successively AMP of60mg·kg-1of body weight once every day for5days, the withdrawal time should be7-day. |
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