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.
Keywords: Chiral column; Fortimicin aminoglycosides; HPLC-ESI–ion trap–MS/MS; Micromonospora olivasterospora; Pentafluoropropionic acid.