title: Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection.
abstract: 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.
authors.affiliation: Center for Intelligent Nano-Bio Materials, Division of Nano Sciences and Department of Chemistry, Ewha Womans University, Seoul 120-750, Korea.
authors.forename: Je Won
authors.initials: JW
authors.lastname: Park
authors.forename: Jay Sung Joong
authors.initials: JS
authors.lastname: Hong
authors.forename: Niranjan
authors.initials: N
authors.lastname: Parajuli
authors.forename: Hwa Soo
authors.initials: HS
authors.lastname: Koh
authors.forename: Sung Ryeol
authors.initials: SR
authors.lastname: Park
authors.forename: Mi-Ok
authors.initials: MO
authors.lastname: Lee
authors.forename: Si-Kyu
authors.initials: SK
authors.lastname: Lim
authors.forename: Yeo Joon
authors.initials: YJ
authors.lastname: Yoon
mesh_terms: D000617:Aminoglycosides / Q000032:analysis / Q000096:biosynthesis; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000096:biosynthesis*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005839:Gentamicins / Q000032:analysis* / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008847:Micromonospora / Q000737:chemistry*; D008970:Molecular Weight; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005839:Gentamicins