高效液相色谱法测定马兜铃酸A在细胞培养基中含量变化*

Determination of the concentration of aristolochic acid A in cell medium with HPLC-UV

关键词:  高效液相色谱法  马兜铃酸A  稳定性

Key words:  HPLC-UV method  aristolochic acid A  stability

摘要:

[目的]建立细胞培养基中马兜铃酸A高效液相色谱(HPLC)法的测定方法,考察在细胞培养条件下马兜铃酸A在培养基中的稳定性。[方法]样品经甲醇沉淀蛋白,10000r/min,离心5min,取上清液进样。采用HPLC法,WatersC¹⁸柱(150nm×4.6nm,5μm),甲醇-水(含3%的冰醋酸)(70:30,V/V)为流动相;检测波长260nm,柱温30℃,流速1mL/min。[结果]马兜铃酸A在0.5~50mg/L范围内线性良好,相关系数r=0.9998;日内、日间精密度相对标准偏差(RSD)分别为0.96%、1.92%;回收率为94.01%~104.34%。[结论]马兜铃酸A在培养基中3d内含量稳定,该法简便、灵敏、特异性强,适用于细胞培养基中马兜铃酸A含量的测定。

Abstract:

[Objective] To establish the HPLC-UV method for determination of the concentration of aristolochic acid A. [Methods] Samples was precipitated by methanol and centrifugated for 5 min (10 000 r/min). Using HPLC-UV method the samples was determined, and the chromatographic system was consisted of Waters C 18 column (150 nm×4.6 nm,5 μm),with 3% acetic acid water solution-methanol (30:70) at the flow rate of 1.0 mL/min. The detection wavelength was 260 nm and column temperature was 30℃. [Results] The aristolochic acid A showed a good linear relationship at the range of 0.5~50 mg/L (r=0.999 8). The intra-run and inter-run validation were 1.92%, 0.96%. The recovery rate were 94.01%~104.34%. [Conclusion] The samples are stabile in 3 days. The method is high specific and sensitive, simple and reliable, so it can be used to study the concentration of aristolochic acid A in culture medium.