Comprehensive Chemical Analysis of Fermentation Extract

An aliquot (1 mg) of the fermentation culture extract was prepared for chemical analysis by filtration over GracePure C18-Max 100 mg/1 mL SPE cartridges (Grace Technologies, USA). Compounds were eluted from the column using acetonitrile and methanol to produce a final volume of 1 mL. This material was taken for nominal mass resolution LC-PDA-MS/MS analysis on a Thermo Finnigan system with a Surveyor PDA Plus Detector, Autosampler Plus, and LC Pump Plus coupled to an LCQ Advantage Plus mass spectrometer (Thermo Fisher Scientific, USA), and with a Phenomenex Kinetex 150 mm × 10 mm × 5 µm C₁₈ analytical column installed (Phenomenex, USA). The UV–Vis spectrum from 200 to 600 nm and positive mode ESI mass spectrum from m/z 190–2000 were recorded, and the mass spectrometer was configured for an automated sample-dependent MS/MS scan. Data were analyzed both manually and by mass spectrometric molecular networking using the Global Natural Products Social Molecular Networking (GNPS) (Wang et al. 2016 (link)). This was performed in duplicate using both the native library searching and analog detection search modes. Additionally, for compound dereplication masses of major components of the mixture were searched manually using MarinLit and the Dictionary of Natural Products databases.