Jiang Su Institute for Food and Drug Control, Nanjing 210008, China.
J Chromatogr A. 2013 Jun 21;1295:90-8. doi: 10.1016/j.chroma.2013.04.059. Epub 2013 Apr 27.
This paper describes the separation of the main component micronomicin from its related substances using a new established liquid chromatographic method with pulsed electrochemical detection (LC-PED). The mobile phase consists of 1 volume of acetonitrile and 99 volumes of an aqueous solution containing 1.25% (v/v) trifluoroacetic acid, 0.025% (v/v) pentafluoropropionic acid and 0.85% (v/v) of 50% sodium hydroxide. The pH of the aqueous solution is adjusted to 2.6 with 0.5 M NaOH. The influence of the different chromatographic parameters on the separation was investigated. A quadruple-potential waveform was used as detection waveform. 0.5 M NaOH was added post column at a flow rate of 0.3 mL/min to raise the pH of detection to at least 12. The LOD and LOQ of micronomicin are 0.08 μg/mL (1.6 ng injected) and 0.25 μg/mL (5 ng injected), respectively. The linearity of micronomicin ranges from 0.25 to 60 μg/mL with a correlation coefficient of 0.9978. Intra-day RSD and inter-day RSD of micronomicin are 0.89% and 0.55%, respectively. This method proved to be robust and is also applicable to a wider number of C18 columns. A number of commercial samples of micronomicin sulfate were analyzed using this method and 18 peaks can be separated from the main component and from each other in one sample. Seven peaks could be identified using reference substances. The chemical structure of two unknown impurities could be characterized by LC-MS based on comparison of their fragmentation patterns with those of available reference substances.
本文描述了使用新建立的带有脉冲电化学检测（LC-PED）的液相色谱法从相关物质中分离主要成分米诺霉素。流动相由 1 份乙腈和 99 份含有 1.25%（v/v）三氟乙酸、0.025%（v/v）五氟丙酸和 0.85%（v/v）50%氢氧化钠的水溶液组成。用水将水溶液的 pH 值调节至 2.6。考察了不同色谱参数对分离的影响。使用四重电位波形作为检测波形。0.5 M NaOH 以 0.3 mL/min 的流速在线添加到柱后，将检测 pH 值提高到至少 12。米诺霉素的检出限和定量限分别为 0.08 μg/mL（1.6 ng 进样）和 0.25 μg/mL（5 ng 进样）。米诺霉素的线性范围为 0.25-60 μg/mL，相关系数为 0.9978。米诺霉素的日内 RSD 和日间 RSD 分别为 0.89%和 0.55%。该方法被证明是稳健的，也适用于更多种类的 C18 柱。使用该方法分析了几种商业米诺霉素硫酸盐样品，在一个样品中可以从主成分和彼此之间分离出 18 个峰。使用对照品可以鉴定出 7 个峰。通过比较其碎片模式与可用对照品的碎片模式，基于 LC-MS 可以对两种未知杂质的化学结构进行表征。
