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 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.
Park
Je Won
JW
Center for Intelligent Nano-Bio Materials, Division of Nano Sciences and Department of Chemistry, Ewha Womans University, Seoul 120-750, Korea.
Hong
Jay Sung Joong
JS
Parajuli
Niranjan
N
Koh
Hwa Soo
HS
Park
Sung Ryeol
SR
Lee
Mi-Ok
MO
Lim
Si-Kyu
SK
Yoon
Yeo Joon
YJ
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
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005839:Gentamicins