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 language | English |
|---|---|
| Pages (from-to) | 4860-4869 |
| Number of pages | 10 |
| Journal | Analytical chemistry |
| Volume | 79 |
| Issue number | 13 |
| DOIs | |
| Publication status | Published - 2007 Jul 1 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection
AU - Park, Je Won
AU - Hong, Jay Sung Joong
AU - Parajuli, Niranjan
AU - Hwa, Soo Koh
AU - Sung, Ryeol Park
AU - Lee, Mi Ok
AU - Lim, Si Kyu
AU - Yeo, Joon Yoon
PY - 2007/7/1
Y1 - 2007/7/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=34447314501&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34447314501&partnerID=8YFLogxK
U2 - 10.1021/ac070028u
DO - 10.1021/ac070028u
M3 - Article
C2 - 17521166
AN - SCOPUS:34447314501
VL - 79
SP - 4860
EP - 4869
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 13
ER -