Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection
Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection
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DOI：
10.1021/ac070028u
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发表时间：
2007-07-01
影响因子：
7.4
通讯作者：
Yoon, Yeo Joon
中科院分区：
文献类型：
--
作者：
Park, Je Won;Hong, Jay Sung Joong;Yoon, Yeo Joon
在本研究中，我们开发了一种灵敏的和高选择性的方法检测的生物合成中间体参与庆大霉素途径的细胞培养的小单孢菌棘孢菌。采用双固相萃取（SPE）技术从细胞培养液中纯化并回收庆大霉素相关成分，并采用高效液相色谱-电喷雾质谱联用（HPLC-ESI-MS/MS）分析庆大霉素中间体的提取物。在提取之前，将培养液的pH调节至pH 2的酸性条件。首先使用反相AccuBOND C-18柱清洗样品，然后使用阳离子交换剂OASIS MCX柱纯化氨基糖苷类组分。发现通过该方法处理的空白培养基中加标的庆大霉素标准品的庆大霉素C复合物的每种组分的检测限约为5 ng，并且当通过HPLC-ESI-MS/MS分析时，标准庆大霉素的每种组分的平均回收率高于91%。主要产生庆大霉素C复合物的棘孢菌ATCC 15835，以及产生庆大霉素B作为主要产物的UV诱导突变株KCTC 10506 BP。除庆大霉素C复合物外，在两种M的培养液中还检测到7种中间体（巴龙胺、庆大霉素A2、B、X2、A、JI-20 A和JI-20 B）。当通过MS/MS分析每种庆大霉素组分的不同裂解模式时，本报告展示了庆大霉素途径中涉及的广泛结构相关生物合成中间体的HPLC分析的第一个例子。
In the present study, we developed a sensitive and highly selective method of detecting the biosynthetic intermediates involved in the gentamicin pathway from a cell culture of Micromonospora echinospora. A novel extraction method utilizing a dual solid-phase extraction (SPE) technique was employed to purify and recover all of the gentamicin-related components from the cell culture broth, and high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS/MS) was used to analyze the extractant for gentamicin intermediates. The pH of the culture broth was adjusted to an acidic condition of pH 2 prior to the extraction. The samples were first cleaned with a reversed-phase AccuBOND C-18 cartridge, and then the aminoglycosidic components were purified using a cationic exchanger OASIS MCX cartridge. The detection limit of a gentamicin standard spiked in blank medium processed by this method was found to be approximately 5 ng for each component of the gentamicin C complex, and the mean recovery for each component of standard gentamicin was above 91% when analyzed by HPLC-ESI-MS/MS. We further demonstrated that this method enables the analytical profiling of the gentamicin-related compounds produced by wild-type M. echinospora ATCC 15835, which mainly produces the gentamicin C complex, and the UV-induced mutant strain KCTC 10506BP, which produces gentamicin B as the major product. Seven intermediates (paromamine, gentamicin A2, B, X2, A, JI-20A, and JI-20B) besides the gentamicin C complex were detected in the culture broth of both M. echinospora strains when analyzed by MS/MS for the distinct fragmentation patterns of each gentamicin component. This report displays the first example of the HPLC profiling in a wide range of structurally related biosynthetic intermediates involved in the gentamicin pathway.