Extraction and Purification of Secondary Metabolites

Secondary metabolites were taken from the culture supernatants extracted using ethyl acetate (Rajan and Kannabiran, 2014 (link)), QuEChERS (Schenck and Hobbs, 2004 (link)), or solid phase extraction (Kamenik et al., 2010 (link)), which was found to be the most suitable and was carried out as follows. An Oasis HLB 3cc 60 mg cartridge (hydrophilic-lipophilic balanced sorbent, Waters, USA) was conditioned with 3 mL methanol (LC-MS grade, Biosolve, Netherlands), equilibrated with 3 mL water (prepared using Milli-Q water purifier, Millipore, USA) and then 3 mL culture supernatant (pH adjusted to 3 with formic acid, 98–100%, Merck, Germany) was loaded. Subsequently, the cartridge was washed with 3 mL water and absorbed substances were eluted with 1.5 mL methanol. The eluent was evaporated to dryness (Concentrator Plus, 2013 model, Eppendorf), reconstituted in 200 μL 50% methanol and centrifuged at 12,000 × g for 5 min.