Characterization of fortimicin aminoglycoside profiles produced from Micromonospora olivasterospora DSM 43868 by high-performance liquid chromatography-electrospray ionization-ion trap-mass spectrometry

Abstract

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were <5% with accuracy in the range from 87.1 to 94.2%. Moreover, the epimeric mixtures including FOR-KH, FOR-KR, and FOR-B were separately resolved through a macrocyclic glycopeptide (teicoplanin)-bonded chiral column. As a result, ten natural FOR pseudodisaccharide analogs were identified and semi-quantified in descending order as follows: FOR-A, FOR-B, DCM, FOR-KH plus FOR-KR, FOR-KK1, FOR-AP, FOR-KL1, FOR-AO, and FOR-FU-10. This is the first report on both the simultaneous characterization of diverse structurally closely related FORs derived from bacterial fermentation using HPLC-ESI-ion trap-MS/MS analysis and the chromatographic separation of the three FOR epimers.

Istamycin aminoglycosides profiling and their characterization in Streptomyces tenjimariensis ATCC 31603 culture using high‐performance liquid chromatography with tandem mass spectrometry

“…Extraction and cleanup protocols for the ISTs in bacterial fermentation were conducted by the slight modification of our recent publication . Whole cultures were transferred into a polypropylene conical tube (BD Biosciences), and the pH was adjusted into ∼2.0 with HCl.…”

“…Analyses of ISTs obtained from the S. tenjimariensis culture were conducted by the slight modification of our recent publication . A Spectra SYSTEM P1000XR quaternary pump in‐lined with an LCQ ion‐trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) was operated.…”

“…In of the five sets of epimeric mixtures ( 1 and 2 ; 5 and 6 ; 7 and 8 ; 11 and 12 ; 15 and 16 ), the analytes were separately identified on an Astec Chirobiotic T column (2.1 × 100 mm, 5.0 μm, Advanced Separation Technologies, Whippany, NJ, USA) using HPLC–MS/MS instruments as previously published . An isocratic mobile phase of 10 mM methanolic ammonium formate was used at a flow rate of 250 μL/min.…”

“…There have been no reports on HPLC profiling of the ISTs biosynthesized during S. tenjimariensis cultivation. Aminoglycosides can be detected using ESI ion trap MS/MS, which provides stronger structural evidence by which to propose the fragmentation pattern of target analyte and its molecular mass . There has been a number of updated chromatographic separation tools developed for aminoglycosides; HILIC has been credited as a useful alternative to the above‐mentioned ion‐pairing chromatography, in which the presence of ion‐pairing reagents may suppress the ionization of the analytes and thus diminish the mass detection efficiency .…”

“…In our most recent publication , we developed an HPLC–ESI‐MS/MS analytical method for the identification of aminocyclitol FOR biosynthetic intermediates produced in Micromonospora olivasterospora DSM 43868. In addition, three kinds of FOR epimer mixtures were chromatographically resolved through a chiral column.…”