基于高效液相色谱法对吸水链霉菌发酵液中波拉霉素A的测定与分离纯化

Determination and purification of polaramycin A from Streptomyces hygroscopicus fermentation broth by HPLC

该研究建立了检测吸水链霉菌(LP-93)发酵液中波拉霉素A含量的HPLC方法,使用C18色谱柱(250 mm×4.6 mm,5μm),确定了流动相[V(乙腈)∶V(水)=45∶55]及相关参数:流速1 mL/min,检测波长220 nm,进样量10μL,柱温30℃。并在此基础上建立了波拉霉素A的高效制备色谱(high performance preparative chromatography,HPPC)分离纯化方法,使用C18色谱柱(250 mm×21.2 mm,7μm),流速15 mL/min,进样量3 mL。进一步使用超高效液相色谱-四极杆-飞行时间串联质谱(ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometer,UPLC-Q-TOF)对纯化组分进行了定性分析。结果表明,该检测方法在2.5~500 mg/L线性关系良好(R^(2)=0.9998),平均回收率在90.53%~101.96%,相对标准偏差(relative standard deviation,RSD)<2%。纯化方法得到较高纯度的波拉霉素A溶液,平均纯度87.52%,平均得率71.84%,并获得波拉霉素A的一级和二级质谱图。该操作方法简便,分析快速,结果准确,可用于吸水链霉菌发酵液中波拉霉素A的含量测定,并为该天然产物的分离纯化提供了参考。

A method for the determination of polaramycin A from Streptomyces hygroscopicus(LP-93)fermentation broth by HPLC was established in this work.C18 column(250 mm×4.6 mm,5μm)and acetonitrile-water(45∶55)mobile phase were employed,as well the related parameters:flow rate of 1.0 mL/min,detection wavelength of 220 nm,injection volume of 10μL,and column temperature of 30℃.On this basis,a high performance preparative chromatography(HPPC)purification method of polaramycin A was also established,in which C18 column(250 mm×21.2 mm,7μm),a flow rate of 15 mL/min and injection volume of 3 mL were employed.Further,the purified fraction was qualitatively analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometer(UPLC-Q-TOF).The results showed that the method exhibited good linearity with a coefficient of determination(R^(2)=0.9998)in the range of 2.5-500 mg/L.The average recoveries of the analyte were between 90.53%-101.96%,with relative standard deviation(RSD)less than 2%.Polaramycin A solution with high purity was obtained,with an average purity of 87.52%and an average yield of 71.84%.MS 1 and MS 2 spectra of polaramycin A were acquired.The method is rapid,simple,and accurate,which can be used for the determination of polaramycin A from S.hygroscopicus fermentation broth,and it provides a reference for the purification of this natural product.