Establishment and Application of Reversed-phase High Performance Liquid Chromatography Method for Detection of Related Substances of Rifaximin Uterine Infusion in Dairy Cows

The study was aimed to establish a method for detection of rifaximin related substances by reversed-phase high-performance liquid chromatography with UV detector. The instrument methods was WondaSil C18-WR 250 mm×4.6 mm 5 μm column, mobile phase was methanol∶acetonitrile∶3.16 g/L ammonium formate (concentrated aqueous ammonia adjusted to pH 7.2)=31.5∶31.5∶37, with detection wavelength 276 nm. Area normalization method was used to calculate the limit of detection, limit of quantification and concentration of rifaximin uterine injectant; The precision of the method, linear correlation and related substances specificity were explored. And the degradation degree of the peak area of the principal components was compared by the destruction of the extreme conditions, the content of the related substances and the separation degree. 40 to 150 μg/mL concentration of rifaximin, peak area and concentration showed a good linear relationship (R²=0.9998), the related substances concentration on peak area linear regression equation was y=23 510x+7 182, within a certain period of time after preparation of acids, alkalis, oxidation, high temperature degradation damage in varying degrees, in the main ingredient content in the sample before the damage was 100.00%, after oxidative damage and heat damage the main component was 13%, acids, alkalis destruction of the main component degradation was about 18%, the main component peak retention time was consistency before and after the destruction. The retention time and resolution of rifaximin and related-substance were good. (The separation between the main component peak and the impurity peaks were greater than 1.5, the degree of separation between the impurity peak and impurity peaks greater than 1.2). The substance detection methods of rifaximin uterine injectants was simple and had specificity and good repeatability, selective and sensitive, which could be used to analyze the related substance of rifaximin uterine injectants.