摘要
目的建立一种基于离线二维高效液相色谱-四级杆飞行时间质谱技术(off-line 2D HPLC-Q/TOF-MS)的多黏菌素E(PME)组分的结构分析方法。方法采用《欧洲药典》10版中的硫酸多黏菌素E的分析方法,经半制备液相色谱分别收集含量超过0.15%的馏分,去除溶剂后,注入液质联用系统。在HPLC-Q/TOF-MS方法中,根据建立的基于高分辨质谱的PME的结构解析策略推导其他未知组分的结构。结果通过离线半制备技术既可以富集含量较低的组分,也可以继续通过第二维色谱进行分离,提高峰容量。经质谱分析,共检测到31个组分。除《欧洲药典》中收载的11个组分外,还检测到包括已知组分如多黏菌素E1(PME1)、多黏菌素E2(PME2)、多黏菌素E4(PME4)、多黏菌素E2-缬氨酸(PME2-Val)的异构体和氨基酸取代产物,如2位苏氨酸转化为丝氨酸的PME组分[PME-Ser(2位)]及脂肪酰基链上的取代产物如3′位羟基化的PME2组分(3′-OHPME2),并首次解析出5个PME的脱水化合物,即2位或10位苏氨酸脱水形成脱氢丁氨酸而产生的PME1或PME2脱水物。结论本研究中的off-line 2D HPLC-Q/TOF-MS可以更全面的评价PME产品中的组分,特别是新型PME脱水物的发现扩展了对PME结构多样性的认识,本方法能够更严格地控制该类产品的质量。
OBJECTIVE To develop an off-line two-dimensional HPLC-Q/TOF-MS method to analyze the components in polymyxin E.METHODS The component with a content of more than 0.15%in polymyxin E was collected respectively according to the analytical method in European Pharmacopoeia(EP)10.0.After evaporating the solvent,each fraction was reinjected into the HPLC-Q/TOF-MS system.The structures of unknown components were deduced based on the structural analysis strategy containing three series of ions obtained from high resolution mass spectrometry.RESULTS The off-line semi-preparation could not only enrich the components with low content,but also separate the components those were not separated in the first dimensional HPLC by the second dimensional HPLC to improve the peak capacity.Thirty-one components were identified by the Q/TOF-MS.Besides the 11 components contained in the EP 10,other components such as isomers of PME1,PME2,PME4 and PME2-Val were detected.Amino acid substitution product of PME2-Ser(2)and fatty acyl chain substitution product of 3′-OH PME2 were also identified.In further,five PME dehydrate components including PME1 and PME2 dehydrate were reported for the first time.The threonine at position 2 or 10 was changed to dehydrobutyric acid in these dehydrate components.CONCLUSION In this study,the off-line 2 D HPLC-Q/TOF-MS can comprehensively evaluate the components in PME.The discovery of new PME dehydrates expands the understanding of the structural diversity of PME.This method has proven extrmely useful for strictly controlling the quality of PME and related polypeptides.
作者
张含智
胡迪
田振华
刘浩
ZHANG Han-zhi;HU Di;TIAN Zhen-hua;LIU Hao(Abiochem Biotechnology Co.,Ltd.,Shanghai 200240,China;Shanghai Institute for Food and Drug Control,Shanghai 201203,China;Shanghai Medical Products Administration,Shanghai 200233,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2022年第24期2118-2126,共9页
Chinese Pharmaceutical Journal
