HPLC-Q-TOF-MS分析乙酰吉他霉素中的组分和杂质

Identification of the components and impurities of acetylkitasamycin by HPLC-Q-TOF-MS

目的采用高效液相色谱-四级杆-飞行时间质谱(HPLC-Q-TOF-MS)技术对乙酰吉他霉素中的主要组分和杂质进行鉴定和分析。方法采用Supelco Discovery C18色谱柱,以0.1mol/L乙酸铵溶液(乙酸调pH值至6.0)-乙腈(40:60)为流动相。在正离子模式下获取高分辨质谱数据,结合已有的大环内酯类抗生素质谱裂解规律,解析组分和杂质的结构。根据提取离子质谱图(EIC)峰面积计算乙酰吉他霉素中已知组分和杂质的相对百分含量。结果分析了乙酰吉他霉素中5个主要组分,并鉴定解析了4大类共16个杂质,分别为2'位去乙酰化物、4位去乙酰化物、3''位乙酰化物、4''位去乙酰化物。结论HPLC-Q-TOF-MS技术可快速、灵敏、准确地鉴定乙酰吉他霉素中的组分和杂质。不同厂家样品中组分和杂质的相对含量差异较大,提示产品质量与生产工艺密切相关。

Objective To identify the components and impurities of acetylkitasamycin by high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS).Methods The separation was performed on a Supelco Discovery C18 column using isocratic mobile phase of 0.1mol/L ammonium acetate solution(adjust to pH 6.0 with acetic acid)–acetonitrile(60:40).Full scan and MS2 experiment were performed under the positive ion mode to acquire high-resolution MS data.The chemical structures of the components and impurities were characterized based on the systematic investigation of macrolide fragmentation pathways.The relative percentage of the known components and impurities was calculated considering the peak areas of extracted ion chromatography(EIC)by the area normalization method.Results The high-resolution MS and MS2 data were used for structural elucidation.Five components in acetylkitasamycin were analyzed.In addition,four kinds of impurities(16 impurities in total)were characterized,namely,2′-deacetyl-acetylkitasamycin,4-deacetyl-acetylkitasamycin,3″-acetyl-acetylkitasamycin,and 4″-deacetyl-acetylkitasamycin.To the best of our knowledge,4-deacetyl-acetylkitasamycin,3″-acetyl-acetylkitasamycin,4″-deacetyl-acetylkitasamycin have not been reported before.Conclusion HPLC-Q-TOF-MS is a rapid,sensitive,accurate method for identification of the components and impurities of acetylkitasamyin.The relative percentage of the components and impurities in different samples varied significantly,indicating the quality of acetylkitasamycin strongly relevant to the synthetic process.