The development of an efficient analytical protocol for the reliable identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is both challenging and essential for further studies into gene-to-metabolite networks. A simple and highly selective method for detecting the biosynthetic intermediates involved in the aminoglycosidic nebramycin pathway of Streptomyces tenebrarius was developed and validated. Cleanup utilizing a solid-phase extraction (OASIS MCX SPE) technique provides a simple and reproducible method for extracting the nebramycin factors from a fermentation broth. The separation of each factor through a reversed-phase C18 column using an ion-pairing reagent allowed the simultaneous profiling of the aminoglycosides. By employing the authentic tobramycin spiked into a blank fermentation medium, the combined use of acid extraction, OASIS MCX SPE cleanup, and HFBA (heptafluorobutyric acid)-mediated chromatographic separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was proven to be sufficiently accurate and reliable to analyze the nebramycin factors produced in a culture broth. The detection limit of tobramycin spiked in the culture broth was approximately 1.8 ng mL-1. The mean recovery ranged from 89 to 92%, the intra- and inter-day precision (RSD) was <6% and their accuracy ranged from 88 to 93%. A total of nine nebramycin factors including apramycin, 6″-O-carbamoylkanamycin B, 6″-O-carbamoyltobramycin, 3′-hydoxyapramycin, tobramycin, kanamycin B, NK-1012-1, nebramine, and neamine were identified. This is the first report on the integrated LC-ESI-MS/MS characterization of a wide range of nebramycin factors from a bacterial fermentation broth.
Bibliographical noteFunding Information:
This study was supported by the Korea Science and Engineering Foundation (KOSEF) NRL Program grant (No. R0A-2008-000-20030-0), the KOSEF grant (M10749000201-07N4900-20110, R11-2005-008-00000-0) funded by the Korean government (MEST), and the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science & Technology, Republic of Korea. J.W.P. gratefully acknowledges both a grant (2009-0073043) from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and a grant (20080401034038) from the Biogreen21 Program, Rural Development Administration.
- Liquid chromatography-tandem mass spectrometry
- Nebramycin factor
- Streptomyces tenebrarius
ASJC Scopus subject areas
- Analytical Chemistry
- Environmental Chemistry