Metabolomic Analysis of S. irregularis

The ethyl acetate fraction of S. irregularis was further subjected to LC-HR-ESI-MS metabolomics analysis using Accela HPLC (Thermo Fisher Scientific, Bremen, Germany) according to a procedure described earlier.^{19,20,22 (link)} The injected volume was 10 μl and the temperature of column was adjusted at 20 °C. The sample was exposed to a gradient chromatographic separation technique at a rate of 300 μl min⁻¹ using purified water [total organic carbon (TOC) was 20 ppb] and acetonitrile, each containing 0.1% formic acid. After that, the elution was carried out with 10% acetonitrile, and then the polarity was gradually increased to 100% acetonitrile within 30 min. This step was followed by an isocratic elution for 5 min preceding a gradient decrease to 10% acetonitrile for 1 min. The HR-ESI-MS results were supplied in both negative and positive ionization modes. The raw data were analyzed using the MZmine 2.12 software to achieve better identification of compounds.^{23 (link)} The detailed steps are illustrated in the ESI Data.†