反相HPLC分析硫氰酸红霉素的相关物质及主组分含量

Determination of Erythromycin Thiocyanate and Its Related Substances by Reverse Phase-High Performance Liquid Chromatography

目的建立一种适用于硫氰酸红霉素相关物质分析及主组分含量测定的质控分析方法。方法采用RP—HPLC，基于硅胶的并耐受高pH值的十八烷基硅烷键合柱为固定相，以水0.01mol·L^-1磷酸氢二钠溶液（pH10．3）-乙腈为流动相；指定以Water公司XTerra@RP18色谱柱进行相关物质分析，流动相比例定为25：35：40；指定以Agilent公司Extend—C18R色谱柱进行主组分含量测定，流动相比例定为18：35：47。流速为1，0mL·min^-1；柱温为50℃；检测波长为210nm。结果硫氰酸红霉素主组分红霉素A色谱峰的理论板数高于10000；红霉素A与最大次组分红霉素E的分离度符合要求。在本实验建立的反相色谱系统中，红霉素对照品的线性范围是0．515～2．161g·L^-1，供试品的线性范围是0.538～2.011g·L^-1；以主成分自身对照法检测供试品中所含次组分、杂质或降解产物，线性范围是0．5％～10％。本法的检测限为10～15mg·L^-1，定量限为60.1mg·L^-1.方法重复性好。结论作为与抗生素微生物检定法并列，但专属性更强，更精密、准确、耐用的色谱分析方法，用于硫氰酸红霉素的相关物质分析与主组分含量测定，可明显提升产品质控水平。

OBJECTIVE To develop a method for the determination of erythromycin thiocyanate and its related substances by RPHPLC ,and investigate the influence factors on chromatographic parameters. METHODS Two high pH stability columns based on silica gel were used respectively （ Agilent Zorbax Extend-C18 column for content determination;and Waters XTerra@ RP18 column for relat- ed substances analysis）. The mobile phase consisted of water -.0.01 mol·L^-1 dibasic sodium phosphate buffer solution （the pH value was adjusted to 10. 3 by sodium hydroxide TS） - acetonitrile （ 18： 35：47 for content determination and 25： 35：40 for related substances analysis）. Both of the two columns were maintained at a constant temperature of about 50 ℃. The flow rate was 1.0 mL per minute. The UV detection wavelength was set at 210 nm. RESULTS The composition of mobile phase showed the greatest influence on retention and selectivity. Erythromycin thiocyanate was relatively stable in mobile phase. The major component, erythromycin A and its related substances were separated and assayed by chromatography. Content determination and related substances analysis all showed the good reproducibility（ n = 9） of this method. The LOD was 10 - 15 mg·L^-1 and the LOQ was 60. 1 mg·L^-1. CONCLUSION This method can be used to analyze erythromycin thiocyanate content and its related substances in order to improve the quality control level for product of erythromycin thiocyanate substance.