阿替洛尔的手性衍生化RP-HPLC法拆分及在鼠肝微粒体中的测定

Resolution and Determination of Enantiomers of Atenolol in Rats Hepatic Microsome by RP-HPLC

目的 :建立阿替洛尔两对映体在鼠肝微粒体中的RP -HPLC测定方法。方法 :从鼠肝微粒体中提取阿替洛尔消旋体 ,采用手性衍生化试剂 2 ,3 ,4,6 -四 -O -乙酰基 - β -D -吡喃葡萄糖异硫氰酸酯 (GITC)对阿替洛尔消旋体进行柱前手性衍生化再以RP -HPLC拆分 ,测定鼠肝微粒体中阿替洛尔各对映体含量。结果 :建立了在碱性条件下从水相中用乙酸乙酯提取阿替洛尔 ,用GITC进行柱前手性衍生化 ,以及用流动相NaH2 PO4缓冲液 -CH3OH -CH3CN (5 0∶2 0∶30 )在ODS柱上拆分的分析方法 ,2 5 4nm波长处检测。阿替洛尔对映体在 1～ 2 0 μg·mL-1范围内呈良好线性关系 ,S (- ) -阿替洛尔 :Y =840 0 76 5X + 141742 8,r =0 9998;R(+ ) -阿替洛尔 :Y =873 15 7 2X + 192 187 5 ,r =0 9998) ,LOD为 5 5ng·mL-1,LOQ为 145ng·mL-1(RSD <7% ) ,相对回收率在 95 %～ 10 5 % (RSD <5 % )。用此方法试验了阿替洛尔在鼠肝微粒体中的代谢测定。结论

Objective:To establish a RP-HPLC method to determine enantiomers of atenolol in rats hepatic microsome.Methods:Extract racemic atenolol from rat hepatic microsome and react with a chiral derivative reagent 2,3,4,6-tetra- O -acetyl-β- D -glucopyranosyl isothiocynate (GITC) to form diastereomeric compounds which are separated with a ODS column.Results:A procedure was set up that atenolol was extracted with ethyl estate from rat hepatic microsome and pre-column derivatized with GITC, the diastereomeric compounds were separated in ODS column with the mobile phase of sodium dihydrogen phosphate buffer-methanol-acetonitrile (50∶20∶30), the detection wavelength was 254 nm. The linear range was 1～20 μg·mL -1 (A-atenolol: Y = 840 076 5 X +141 742 8, r =0 999 8;B-atenolol: Y =873 157 2 X + 192 187 5 , r =0 999 8 ). LOD tested was 110 ng·mL -1 and LOQ was 290 ng·mL -1 ( RSD <7%). The relative recovery at different concentration were ranged from 94% to 105%。The method was practiced to observe the concentration variance of atenolol enantiomers in rat hepatic microsome after different incubation times.Conclusion:A practical determination method of atenolol enantiomers in rat hepatic microsome by pre-column chiral derivatization RP-HPLC was established. The procedure can be employed to study the enzymatic behavior of atenolol.