HPLC法测定发酵液中丰加霉素的研究

Identification and quantification of toyocamycin in fermentation broth by HPLC

丰加霉素是核苷类抗生素的一种,多由链霉菌发酵产生.本研究建立了放线菌发酵液中丰加霉素的高效液相色谱检测方法,采用SHIMADZU C18色谱柱(150mm×4.6mm,5μm),甲醇为流动相A,水为流动相B,梯度洗脱,洗脱程序为0～15min,5%～37%A;15～30min,37%～100%A;30～40min,100%～5%A;流速1.0mL·min-1,检测波长279nm,柱温30℃.该色谱条件下,丰加霉素能得到良好的分离,定量法的线性范围为5～150mg·L-1(r=0.999 7),平均加样回收率为101.01%,RSD(%)为0.62.本测定方法为今后高产丰加霉素的菌种选育和发酵工艺等研究提供了一套准确的检测技术.

Toyocamycin,one of nucleoside antibiotics,is mostly biosynthesized by Streptomyces genera.So far,little is known about the determination method of toyocamycin in fermentation broth from actinomycetes.The identification and quantification method of toyocamycin by HPLC was established.The separation was performed on C18 column(150mm×4.6mm,5μm,Shimadzu).The mobile phase consisted of water(A)and methanol(B).The gradient elution program was:0～15min,5%～37% A,15～30min,37%～100% A and 30～40min,100%～5% A.The flow rate was 1.0mL·min-1.The detection wavelength was 279nm.The column temperature was 30 ℃.Toyocamycin was completely separated by HPLC,and the calibration curve was linear within the range of 5～150mg·L-1(r=0.999 7).The average recovery of toyocamycin was 101.01%and RSD was 0.62%.The determination method will facilitate further study on strain breeding and fermentation condition optimization.