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

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 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
Volume79
Issue number13
DOIs
Publication statusPublished - 2007 Jul 1
Externally publishedYes

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Electrospray ionization
High performance liquid chromatography
Gentamicins
Cell culture
Aminoglycosides
Mass spectrometry
Recovery

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection. / Park, Je Won; Hong, Jay Sung Joong; Parajuli, Niranjan; Hwa, Soo Koh; Sung, Ryeol Park; Lee, Mi Ok; Lim, Si Kyu; Yeo, Joon Yoon.

In: Analytical chemistry, Vol. 79, No. 13, 01.07.2007, p. 4860-4869.

Research output: Contribution to journalArticle

Park, Je Won ; Hong, Jay Sung Joong ; Parajuli, Niranjan ; Hwa, Soo Koh ; Sung, Ryeol Park ; Lee, Mi Ok ; Lim, Si Kyu ; Yeo, Joon Yoon. / Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection. In: Analytical chemistry. 2007 ; Vol. 79, No. 13. pp. 4860-4869.
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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 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.",
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AU - Parajuli, Niranjan

AU - Hwa, Soo Koh

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