伊短菌素A标品的制备及HPLC定量分析

Preparation of edeine A standard and its quantitative determination by HPLC

目的制备伊短菌素A标品并建立其定量分析方法。方法发酵液经过预处理后,采用阳离子吸附树脂富集和反相高效液相色谱(RP-HPLC)分离纯化,制备得到伊短菌素A。通过质谱、核磁共振谱等确证化学结构,高效液相色谱法测定样品纯度。定量分析方法采用XAqua C_(18)(250 mm×4.6 mm,5μm)色谱柱,流动相:0.1%TFA水溶液-甲醇(90:10,V/V);流速:1 mL/min,检测波长:272 nm,柱温:35℃,进样量:20μL。结果制备了高纯度的伊短菌素A标品。优化定量条件下伊短菌素A在15~1200 mg/L范围内浓度与峰面积线性关系良好;加标回收率在92.61%~107.33%;测定峰面积的相对标准偏差为0.163566%(n=6),<1.0%;检测限和定量限分别为16.74 ng和55.79 ng。结论本文所建立的制备方法操作简单,易放大生产。定量方法简单快速,准确度和重现性良好。适用于伊短菌素A的定量分析。

Objective To prepare the reference substance of edeine A and establish its quantitative analysis method.Methods After the fermentation broth was pretreated,edeine A was enriched by cationic adsorbent resin and separated and purified by reversed-phase high performance liquid chromatography(RP-HPLC).The chemical structure was confirmed by MS and NMR,and the purity of the sample was determined by HPLC.The quantitative analysis was performed on an Agilent Zorbax Eclipse C_(18) column(250 mm×4.6 mm,5μm)with mobile phase consisting of 0.1%TFA and methanol(90:10,V/V).The flow rate was 1 mL/min,the detection wavelength was set at 272 nm,the column temperature was 35℃,and the injection volume was 20μL.Results The high-purity edine A was prepared.Under the optimized conditions,there was good linear relationship between the concentration and the peak area of edeine A in the range of 15~1200 mg/L.The average recoveries at three levels ranged from 92.61%to 107.33%.The relative standard deviations(RSDs)of peak area was 0.163566%(n=6),<1.0%.The limit of detection and the limit of quantification of edeine A were 16.74 ng and 55.79 ng respectively.Conclusion The method is simple and easy to scale up.The results of the experiments have demonstrated that the established method is rapid and simple with good accuracy and reproducibility.The method is suitable for the quantitative analysis of edeine A.