喹烯酮在草鱼血浆中蛋白结合率的测定

Determination of protein binding of Quinocetone with grass carp(Ctenopharyngodon idellus) plasma

为了研究喹烯酮(Quinocetone,QCT)与草鱼(Ctenopharyngodon idellus)血浆蛋白的结合情况,实验采用超高效液相色谱仪(UPLC)法测定透析袋两侧溶液中喹烯酮的质量浓度,计算QCT的血浆蛋白结合率。血浆样品采用乙腈提取,正己烷脱脂净化。采用ACQUITY UPLC BHE C18分离柱,柱温为30℃,流动相为乙腈-水(30∶70),流速为0.3 mL/min,紫外检测波长为320 nm。本方法的线性范围为0.025~3.0μg/mL,相关指数r2≥0.998。添加水平为0.05、0.1、0.2μg/mL时,平均回收率75%~90%,相对标准偏差均低于15%。结果显示:QCT在质量浓度为0.05、0.2、1μg/mL时与血浆蛋白结合率分别为(81.40±0.80)%、(79.60±0.80)%、(80.54±0.30)%,达到平衡的时间为36 h。喹烯酮的血浆蛋白结合无浓度依赖性,且具有高强度的血浆蛋白结合率。

In order to understand the protein binding of Quinocetone （QCT） with grass carp plasma, a method of ultra- performance liquid chromatography had been developed for determination of the protein binding of Quinocetone with grass carp plasma. After extraction with acetonitrile and defatted with n - hexane, the analytes was separated on ACQUITY UPLC BHE C18 with acetonitrile -water as mobile phase at flow rate of 0. 3 mL/min and column temperature of 30 ℃. The cali- bration curve ranging from 0. 025 - 3.0 μg/mL in fluent phase solvent was used to establish instrument response, with rel- ative standard coefficient beyond 0. 998. The method was validated by analysis of nine replicate plasma samples fortified with three concentration levels of 0. 05 p^g,/L, 0. 1 p^g/L and 0. 2 p,g/L QCT, respectively. Depending on the biological matrix and analyte, relative standard deviations were below 15 % , with average recovery rates around 75 % to 90 % in spiked samples. Finally, the method was successfully used to determine the protein binding of common carp plasma with QCT un- der 4 ~C by equilibrium dialysis method, and the equilibrium time was 36 h and the rate of protein binding were （81.40 ± 0. 8 ） %, （ 79. 60 ± 0. 8 ） %, （ 80. 54 ± 0. 3 ） %, respectively. The protein binding rates of the compounds in the QCT were independent on the area of the concentration which was determinate and the protein binding rates were strength.