HPLC-荧光检测法测定犬血浆中阿德福韦的浓度

Determination of PMEA in beagle dog plasma by HPLC with fluorescence detection

目的:建立HPLC-荧光法测定beagle犬血浆中阿德福韦酯类似物M1、M2的代谢物阿德福韦(Adfovir,PMEA)浓度的方法。方法:色谱柱,C18(150×4.6 mm,5μm);流动相A,含2%乙腈的25 mmol/L磷酸盐缓冲溶液(5 mmol/L氢氧化四丁基铵,pH 6.0);流动相B,含65%乙腈的25 mmol/L磷酸盐缓冲溶液(5 mmol/L氢氧化四丁基铵,pH 6.0);流速,1.0 ml/min;荧光检测,激发波长为236 nm,发射波长为420 nm;柱温,35℃;进样量1,0μl。结果:PMEA血浆浓度在5.0～750.0 ng/ml范围内r(=0.999 8),线性关系良好,最低检测限为2.0 ng/ml。血浆中PMEA的相对回收率在98.36%～99.58%,提取回收率在79.76%～82.14%,日内RSD在2.12%～3.77%和日间RSD在3.07%～5.96%。结论:本方法准确、可靠、重现性好,适用于beagle犬血浆中PMEA的含量测定。

Objective：To establish a method of HPLC-Fluorescence to determine beagle dog plasma concentration of Adefovir,parent drug of analogue of Adefovir dipivoxil-M1,M2.Methods：The analyte was separated on a Benetnach C18 column（150×4.6 mm,5 μm）.The mobile phase were：A,2% acetonitrile in 25 mmol potassium phosphate buffer with 5 mmol tetrabutylammonium hydroxide,pH 6.0;B,65% acetonitrile in 25 mmol potassium phosphate buffer with 5 mmol tetrabutylammonium hydroxide,pH 6.0.The flow rate was 1.0 ml/min and the column temperature was maintained at 35℃.The injection volume was 10 μl.Results：The calibration curve was linear over plasma concentration range of 5.0～750.0 ng/ml,r=0.999 8.The lower limit of quantification in plasma of beagle dogs was 2.0 ng/ml.Relative recovery of Adefovir in plasma of beagle dogs was 98.36%～99.58%,and absolute recovery was 79.76%～82.14%.The interday precision（RSD%） in plasma was 2.12%～3.77%,and the intraday precision（RSD%） was 3.07%～5.96%.Conclusion：The established method has a good selectivity and reproducibility,and is suitable for the determination of Adefovir in plasma of beagle dogs.