高效液相色谱法测定大鼠血清中染料木黄酮浓度

Determination of genistein in rats by HPLC

目的:建立高效液相色谱法测定大鼠血清中染料木黄酮浓度.方法:血清样品用叔丁基甲醚萃取,萃取物用氮气吹干后,用乙腈-0.05 mol·L-1甲酸胺缓冲液(27:73,pH 4.0)溶解用于色谱分析.色谱务件:采用Thermo C18柱(4.6 mm×250 mm,5μm);以乙腈-0.05 mol·L-1甲酸胺缓冲液(27:73,pH 4.0)为流动相;流速为1.0 mL·min-1;检测波长为260 nm;柱温为25℃;进样量为10μL.结果:染料木黄酮最低检测浓度为0.01 mg·L-1;血清样品浓度在0.01～10.00 mg·L-1范围内呈良好的线性关系,回归方程为:y=0.079 8+6.976 4X,r=0.999 2(n=5);相对回收率为(100.1±1.8)%;日内RSD与日间RSD均小于10.00%(n=5).结论:该方法灵敏,简便、快速、重复性好,适用于大鼠血药浓度测定及药代动力学研究.

OBJECTIVE A simple assay method for genistein in rats by high performance liquid chromatography was established. METHODS Serum sample was extracted with tert.-butyl methyl ether(TBME) for HPLC analysis.The HPLC assay was conducted using Thermo C_ 18 colum( 4.6 mm×250 mm,5 μm) with acetonitrile- 0.05 M ammonium formate buffer(27∶73) as mobile phase at a flow-rate of 1.0 mL·min -1 .10 μL of sample solution were injected into the C_ 18 colum where the temperature was 25 ℃ .The detection wavelength was 260 nm. RESULTS The limit of detection for genistein was 0.01 mg·L -1 in serum sample.The linearity range for genistein was obtained from 0.01 mg·L -1 to 10.00 mg·L -1 .The linear regression equation was Y = 0.079 8 + 6.976 4 X , r = 0.999 2 ( n =5).The recoveries of genistein were ( 100.1 ± 1.8 )%.The relative standard deviations of the intra-day and inter-day were less than 10%( n =5). CONCLUSION The method is sensive,simple,rapid,and accurate,which may be used for blood concentration monitoring and pharmacokinetics study for genistein.