Abstract
In terms of risk assessment especially for the impurities with different ultraviolet (UV) absorptions in 16-membered macrolides produced by fermentation, quantification without the availability of corresponding reference substances currently poses a challenge. In this study, a reliable method was established for the analysis of impurities in 16-membered macrolides for the first time by high performance liquid chromatography tandem with charged aerosol detector (HPLC-CAD). The chromatographic conditions and CAD parameters were optimized for a good separation and sensitivity. The impurities were identified by ion trap time-of-flight mass spectrometry (IT-TOF/MS), the developed method was validated, and the sources of degradation impurities in production were investigated. Based on the experimental results, the superiority of the developed method over UV detection was demonstrated that the method provided a universal response to impurities with differences chromophores. In the method validation, good linearity was obtained with coefficient of determination (R2) greater than 0.999 in the range of 2–100 μg·mL−1. The limit of detection (LOD) and limit of quantification (LOQ) were 5 and 15 ng for impurity Ⅲ, respectively. The average content of the impurity Ⅲ was found to be 0.4 % in leucomycin. The recoveries were 98.7 %–102.5 % at the spiked concentration levels of 0.40 %, 0.60 % and 0.80 % with relative standard deviations (RSDs, n=3) lower than 2.0 %. The impurities in 10 leucomycin samples were determined to be 3.2 %–5.2 %. Finally, the acidity of solution during the purification and the temperature of drying process were found to be the main factors that causing the increased amount of impurities. Overall, the developed HPLC-CAD quantification method was a suitable alternative for the analysis of impurities in the 16-membered macrolides discussed in this manuscript. Our study provided guidance for pharmaceutical companies to improve the manufacturing process and control the impurities.
Published Version
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