罗红霉素在中华鳖体内残留检测及药动学研究
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摘要
中华鳖是我国特有的名特水产养殖品种之一,营养丰富,经济价值高,目前已在全国各地广泛养殖。罗红霉素作为第二代大环内酯类广谱抗生素,主要应用于水产养殖中弧菌病、龟鳖白眼病等防治。本文以中华鳖为实验材料,利用液相色谱-串联质谱技术,研究了罗红霉素的快速准确高效检测方法及其在中华鳖体内的代谢规律,旨在为罗红霉素的安全规范用药及质量安全评价提供技术支撑。
1、建立了中华鳖血液中罗红霉素的超高效液相色谱-串联质谱检测方法。优化了检测条件,并比较分析了乙酸乙酯、乙醚、正己烷：异丙醇=95：5(v：v)、乙酸乙酯：乙醚=30：70(v：v)、乙酸乙酯：乙醚=50：50(v：v)、乙酸乙酯：乙醚=50：50(v：v)等六种不同提取剂的提取效果,结果表明以乙酸乙酯为提取液提取效率最高。对乙酸乙酯提取液在五种不同pH条件下的提取效果进行了比较,以pH=8.5的条件下为最好。优化建立的测定方法为：中华鳖血液样品采用乙酸乙酯提取,用碳酸钠溶液调节pH=8.5,正己烷脱脂后,过0.2μm滤膜至进样瓶供液质测定,外标法定量。电喷雾ESI正离子扫描模式下进行多反应MRM监测,选用的离子对为m/z837.6/158(定量用)和837.6/680。罗红霉素的保留时间为1.86min,方法的线性范围为0.1-1000ng/mL,相关系数R=0.9996。在加标浓度5ng/mL-500ng/mL之间,方法的回收率为70.97%-95.46%,相对标准偏差3.90%-9.75%,定量限为0.1n/mL
2、评定了方法的不确定度。建立了血液中罗红霉素残留测定方法的不确定度数学模型,分析了不确定度的主要来源并进行不确定度分量评定。结果表明：方法的不确定度来源包括取样时引入的不确定度,定容体积引入的不确定度以及上机液浓度测定引入的不确定度。对5ng/mL的加标样品进行了六次测定,测定的平均值为4.77ng/mL,合成不确定度为0.2634ng/mL,在95%的置信水平下,其扩展不确定度为0.5268ng/mL
3、研究了罗红霉素在中华鳖体内的残留代谢规律。连续7d投喂含有60mg/kg罗红霉索的药饵,利用液相色谱-串联质谱法分析了肝脏、血液和肌肉中罗红霉素的残留量,并利用3P37药动学软件进行数据分析。结果表明：罗红霉索在中华鳖血液、肌肉和肝脏中达峰时间分别为2h、8h和8h,对应的浓度分别为：19.03μg/kg、5.130μg/kg和13.98μg/kg,药动学方程分别为：C血浆=40.21×e-0.8514t+1.471×e-0.1798t,C肌肉=3.349×e-00816t+5.358×e-0.04430t和C肝脏=4.942×e-0.07221t+0.7362×e-0.006898t,消除除半衰期分别为38.5h、15.6h和100.5h。在此剂量下的建议休药期375度日。
4、初步分析了罗红霉素在中华鳖血液内的代谢产物。利用液相色谱串联质谱技术,比较分析了给药后2h和24h后中华鳖血液中的罗红霉素代谢产物并对其进行了二级质谱分析。结果表明：罗红霉索在中华鳖血液中的代谢产物有三种,分别是N-去甲基代谢物、红霉素肟和罗红霉素脱克拉定糖代谢物,并探讨了罗红霉素的代谢途径。
By using liquid chromatography-tandem mass spectrometry technology, a study on an accurate and efficient detection method of Roxithromycin and its application in the pharmacokinetics of soft-shell turtles were carried out for the evaluation of the quality and safety.
1. An ultra high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the determination of roxithromycin in soft-shell turtle was developed and the detection conditions were optimized. Six different kinds of extractants and five different kinds of extract pH were compared according to the extraction effect. Samples were extracted by ethyl acetate. pH value was adjusted to8.5by sodium carbonate and free lipid was eliminated by hexane. The limit of quantitation (LOQ) for roxithromycin was found to be0.1ng/mL. Under the conditions mentioned above, recoveries ranged from70.97to95.46%at levels of5to500ng/mL with relative standard deviation (RSD) were from3.90%to9.75%.
2. The uncertainty of the method of determination of Roxithromycin was investigated. It was found that the sources of uncertainty consisted of sampling, reconstitution and concentration determination. The uncertainty components and combined uncertainty were calculated. Under the spiked levels of5ng/ml, the combined uncertainty, expanded uncertainty and test result of method were0.2634ng/mL,0.5268ng/mL and4.77±0.5268ng/mL respectively.
3. Pharmacokinetics of roxithromycin were studied in plasma, liver and muscle of soft-shell turtles. After oral administration at a dose of60mg/kg by7consecutive days of feeding, samples were taken at1h,2h,4h,8h,28h,48h,72h,120h after last feeding. The concentrations of roxithromycin were determined by high performance liquid chromatography (HPLC-MS/MS). The results showed that the peak times of concentrations in plasma, muscles, livers were2h,8h,8h with the peak concentrations of19.03μg/kg,5.130μg/kg,13.98μg/kg, respectively. According to3P97analysis, roxithromycin in plasma, liver and muscle followed two compartment model. Equations were as follows:
Cplasma=40.21xe-0.8514t+1.471xe-0.1798t;
Cmusclc=3.349xe-0.0816t+5.358xe-0.04430t;
Clivcr=4.942xe-0.07221t+0.7362xe-0.006898t,
The elimination half-life (T(1/2)β) of roxithromycin in plasma, muscle, liver in soft-shell turtle were38.55h,15.65h and100.5h and recommended withdrawal period was15days under the water temperature of25±2℃
4. The metabolites and metabolic pathway of roxithromycin in the plasma in soft-shell turtle at2h and after24h after feeding were analyzed by mass spectrometry analysis. The results showed that two metabolites (erythromycin-oxime, N-mono-and didemethylated derivatives of roxithromycin) were detected after2h and3metabolites (erythromycin-oxime, N-mono-and didemethylated derivatives of roxithromycin and deseladinose-roxithromycin) were detected after24h.
1、建立了中华鳖血液中罗红霉素的超高效液相色谱-串联质谱检测方法。优化了检测条件,并比较分析了乙酸乙酯、乙醚、正己烷：异丙醇=95：5(v：v)、乙酸乙酯：乙醚=30：70(v：v)、乙酸乙酯：乙醚=50：50(v：v)、乙酸乙酯：乙醚=50：50(v：v)等六种不同提取剂的提取效果,结果表明以乙酸乙酯为提取液提取效率最高。对乙酸乙酯提取液在五种不同pH条件下的提取效果进行了比较,以pH=8.5的条件下为最好。优化建立的测定方法为：中华鳖血液样品采用乙酸乙酯提取,用碳酸钠溶液调节pH=8.5,正己烷脱脂后,过0.2μm滤膜至进样瓶供液质测定,外标法定量。电喷雾ESI正离子扫描模式下进行多反应MRM监测,选用的离子对为m/z837.6/158(定量用)和837.6/680。罗红霉素的保留时间为1.86min,方法的线性范围为0.1-1000ng/mL,相关系数R=0.9996。在加标浓度5ng/mL-500ng/mL之间,方法的回收率为70.97%-95.46%,相对标准偏差3.90%-9.75%,定量限为0.1n/mL
2、评定了方法的不确定度。建立了血液中罗红霉素残留测定方法的不确定度数学模型,分析了不确定度的主要来源并进行不确定度分量评定。结果表明：方法的不确定度来源包括取样时引入的不确定度,定容体积引入的不确定度以及上机液浓度测定引入的不确定度。对5ng/mL的加标样品进行了六次测定,测定的平均值为4.77ng/mL,合成不确定度为0.2634ng/mL,在95%的置信水平下,其扩展不确定度为0.5268ng/mL
3、研究了罗红霉素在中华鳖体内的残留代谢规律。连续7d投喂含有60mg/kg罗红霉索的药饵,利用液相色谱-串联质谱法分析了肝脏、血液和肌肉中罗红霉素的残留量,并利用3P37药动学软件进行数据分析。结果表明：罗红霉索在中华鳖血液、肌肉和肝脏中达峰时间分别为2h、8h和8h,对应的浓度分别为：19.03μg/kg、5.130μg/kg和13.98μg/kg,药动学方程分别为：C血浆=40.21×e-0.8514t+1.471×e-0.1798t,C肌肉=3.349×e-00816t+5.358×e-0.04430t和C肝脏=4.942×e-0.07221t+0.7362×e-0.006898t,消除除半衰期分别为38.5h、15.6h和100.5h。在此剂量下的建议休药期375度日。
4、初步分析了罗红霉素在中华鳖血液内的代谢产物。利用液相色谱串联质谱技术,比较分析了给药后2h和24h后中华鳖血液中的罗红霉素代谢产物并对其进行了二级质谱分析。结果表明：罗红霉索在中华鳖血液中的代谢产物有三种,分别是N-去甲基代谢物、红霉素肟和罗红霉素脱克拉定糖代谢物,并探讨了罗红霉素的代谢途径。
By using liquid chromatography-tandem mass spectrometry technology, a study on an accurate and efficient detection method of Roxithromycin and its application in the pharmacokinetics of soft-shell turtles were carried out for the evaluation of the quality and safety.
1. An ultra high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the determination of roxithromycin in soft-shell turtle was developed and the detection conditions were optimized. Six different kinds of extractants and five different kinds of extract pH were compared according to the extraction effect. Samples were extracted by ethyl acetate. pH value was adjusted to8.5by sodium carbonate and free lipid was eliminated by hexane. The limit of quantitation (LOQ) for roxithromycin was found to be0.1ng/mL. Under the conditions mentioned above, recoveries ranged from70.97to95.46%at levels of5to500ng/mL with relative standard deviation (RSD) were from3.90%to9.75%.
2. The uncertainty of the method of determination of Roxithromycin was investigated. It was found that the sources of uncertainty consisted of sampling, reconstitution and concentration determination. The uncertainty components and combined uncertainty were calculated. Under the spiked levels of5ng/ml, the combined uncertainty, expanded uncertainty and test result of method were0.2634ng/mL,0.5268ng/mL and4.77±0.5268ng/mL respectively.
3. Pharmacokinetics of roxithromycin were studied in plasma, liver and muscle of soft-shell turtles. After oral administration at a dose of60mg/kg by7consecutive days of feeding, samples were taken at1h,2h,4h,8h,28h,48h,72h,120h after last feeding. The concentrations of roxithromycin were determined by high performance liquid chromatography (HPLC-MS/MS). The results showed that the peak times of concentrations in plasma, muscles, livers were2h,8h,8h with the peak concentrations of19.03μg/kg,5.130μg/kg,13.98μg/kg, respectively. According to3P97analysis, roxithromycin in plasma, liver and muscle followed two compartment model. Equations were as follows:
Cplasma=40.21xe-0.8514t+1.471xe-0.1798t;
Cmusclc=3.349xe-0.0816t+5.358xe-0.04430t;
Clivcr=4.942xe-0.07221t+0.7362xe-0.006898t,
The elimination half-life (T(1/2)β) of roxithromycin in plasma, muscle, liver in soft-shell turtle were38.55h,15.65h and100.5h and recommended withdrawal period was15days under the water temperature of25±2℃
4. The metabolites and metabolic pathway of roxithromycin in the plasma in soft-shell turtle at2h and after24h after feeding were analyzed by mass spectrometry analysis. The results showed that two metabolites (erythromycin-oxime, N-mono-and didemethylated derivatives of roxithromycin) were detected after2h and3metabolites (erythromycin-oxime, N-mono-and didemethylated derivatives of roxithromycin and deseladinose-roxithromycin) were detected after24h.
引文
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