Separation Of Bitespiramycin Components By HSCCC

Bitespiramycin, a novel antibiotic produced by genetically engineered strain, has higher biological activity than spiramycin. The major components of bitespiramycin are isovalerylspiramycinⅠ,Ⅱ,Ⅲ, therefore, preparative separation of these three components is significant to medcine quality control.Bitespiramycin has multi-components which are similar to each other on chemical structure and properties, therefore, a separation method with high distinguishability and efficiency is needed. High speed counter-current chromatography (HSCCC) is a new separation method based on liquid-liquide distibution, and provides a highly efficent and convenient way for preparative separation of isovalerylspiramycin. This paper is a sthdy on the separation of isovalerylspiramycinⅠ,Ⅱ,Ⅲfrom its bitespiramycin complex by HSCCC.Selection and optimization of the two-phase solvent system is of great importance for this chromatographic method. Hence, as the first step, the K value of isovalerypirmycins in a series of solvent systems and their setting time, important elements for selection, was investigated in detail, and five systems suitable for HSCCC separation were gained. Furthermore, effect of temperature and pH on K value and setting time was studied, set n-hexane-ethyl acetate-ethanol-water at volume ratio of 10:1:5:7 as an example, and the optimized condition was 25℃, without ajustment of pH.Then the five systems were applied to semi-preparative (separation column is 300 mL) HSCCC separation. It was indicated that n-hexane-ethyl acetate-ethanol-water at volume ratio of 10:1:5:7 has the best separating effect, considerating the purity and yield of isovalerylspiramycin products. With the selected system, separating conditions, such as flow rate of mobile phase, time for collecting and quantity of sample, were optimized. As a result, a way of gradient elution and a injection quantity of 125mg soluble in 20mL mobile phase were adopted and the purificantion of isovalerylspiramycinⅡandⅢgained through separation reached 98.1% and 99.1%, respectively. Besides, the purificantion of isovalerylspiramycin I was about 40% and could be used for further purification.Last but not least, preparative separation of isovalerylspiramycinⅡ,Ⅲwas carried out by preparative (separation column is 1000 mL) HSCCC, based on the experiments of semi-preparative HSCCC separation. When the injection quantity was 400mg soluble in 80mL mobile phase, isovalerylspiramycin II gained through one separation reached 40mg, with a purification above 90%, and isovalerylspiramycin III reached 50mg, with a purification above 97%. Mass analsis was then made for the isovalerylspiramycins product, and the chemical valence and relations between mass concentration in solution and chromatographic peak areas were tested, which provided references for the quality control of bitespiramycin.