Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection

Je Won Park, Jay Sung Joong Hong, Niranjan Parajuli, Soo Koh Hwa, Ryeol Park Sung, Mi Ok Lee, Si Kyu Lim, Joon Yoon Yeo

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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 C18 cartridge, and then the aminoglycoside 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.

Original languageEnglish
Pages (from-to)4860-4869
Number of pages10
JournalAnalytical chemistry
Issue number13
Publication statusPublished - 2007 Jul 1
Externally publishedYes


ASJC Scopus subject areas

  • Analytical Chemistry

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