液相色谱串联质谱法测定兔眼玻璃体内二十碳五烯酸的浓度

目的:建立适用于兔眼玻璃体内二十碳五烯酸(eicosapentaenoic acid,EPA)浓度测定的LC-MS/MS方法,为研究EPA在玻璃体内的药动学提供技术支持。方法:应用Q Trap 4500液质联用仪,采用电喷雾电离源,在负离子方式选择反应监测(MRM)模式下测定兔眼玻璃体内注射EPA后不同时间的药物浓度。50μL玻璃体样品经乙腈沉淀蛋白并浓缩处理后用于定量分析。色谱分离采用XDB C₁₈柱(50 mm×4.6 mm,1.8μm),流动相为甲醇-水-甲酸(90∶10∶0.1,V/V/V),流速:1.00mL·min-1;进样量:2μL。监测的离子反应分别为m/z301.0→m/z257.3(EPA)和m/z526.0→m/z401.0(内标,格列喹酮)。每个样品的分析时间为3 min。结果:玻璃体内EPA在0.02~5.00mg·L^-1范围内线性关系良好,最低定量限为0.02mg·L^-1;提取回收率大于90%;日内和日间精密度(RSD)均小于11.6%;基质效应可忽略;EPA玻璃体样品室温放置6 h、-20℃放置3 d、-70℃一次冻融稳定,处理后样品室温放置20 h稳定。兔眼内注射EPA后的主要药动学参数如下:Cmax为1.05 mg·L^-1,tmax为0.5 h,t_1/2为4.72 h,AUC(0-t)为3.614 mg·L^-1·h,AUC(0-∞)为4.296 mg·L^-1·h。结论:所建立的EPA浓度测定的LC-MS/MS方法灵敏、准确、简便、稳定,适用于EPA在兔眼内的药动学研究。

Determination of eicosapentaenoic acid in rabbit vitreous by LC-MS/MS method

OBJECTIVE To establish an applicable LC-MS/MS method for the determination of eicosapentaenoic acid (EPA) in rabbit vitreous.METHODS Qtrap 4500 tandem quadrupole mass spectrometer equipped with Turbo Spray source was used to determine the concentration of EPA at designed time after injection of EPA in rabbit vitreous.50 μL of rabbit vitreous sample was used for analysis after protein precipitation and concentration.Separation was conducted on a XDB C₁₈ column (50 mm×4.6 mm,1.8 μm) using acetonitrile-water-formic acid (90:10:0.1,V/V/V) as the mobile phase.The flow rate was 1.0 mL/min,the column temperature was 40℃ and the injection volume was 2 μL.Analysts were detected by multiple reaction monitoring (MRM) at negative ion mode.The precursors to product ion combinations of m/z301.0→m/z257.3 were used for quantification of EPA,and m/z526.0→m/z401.0 for gliquidone (internal standard).The overall running time of a single sample was 3.0 min.RESULTS The linear concentration range of EPA was 0.02 to 5.00 mg·L^-1,the low limit of quantification was 0.02 mg·L^-1.Extraction recovery was more than 90%.Intra-day and inter-day RSD values were both less than 11.6%.Matrix effect was negligible.Samples were stable at room temperature for 6 h,-20℃ for 3 d at the least and the processed samples were stable at room temperature for 20 h.The main pharmacokinetic parameters of EPA in rabbit vitreous were as follows:C_max 1.05 mg·L^-1,t_max 0.5 h,t_1/24.72 h,AUC_(0-t) 3.614 mg·L^-1·h,AUC_(0-∞) 4.296 mg·L^-1·h.CONCLUSION The developed LC-MS/MS method for the determination of EPA is sensitive,accurate,simple and reproducible.It is applicable for the pharmacokinetic research of EPA in rabbit vitreous.