GNPS Colostethus imbricolus - SBC18 column

All analysis were performed in reversed-phase HPLC Zorbax SB-C18 column (150 mm x 2.0 mm x 3.0 um, Agilent technologies, Santa Clara, USA) for optimizing the separation conditions. The mobile phases were A (0.1% v/v formic acid in Milli-Q water) and B (0.1% v/v formic acid in ACN) at a flow rate of 0.3 mL/min and 45ºC. Gradient elution started to 5%B for 2 min, then increased to 20%B at 4 min, then to 30%B at 15 min, 34%B at 25 min, 45%B at 30, and finally to 100%B at 45 min and maintained for 5 min and thereafter returned back to starting conditions in 2 min and finally 13 min of re-equilibration time was applied. Total run time was 65 min. MS spectra were acquired in positive ion mode in the range of 20–2000 m/z and a scan rate of 6 scan/min.
10 uL of each frog extract was injected by duplicate using an the same HPLC-Q-TOF system (Agilent Technologies, Santa Clara, CA, USA) employed for the targeted analysis. Mass range for the full scan was performed between 20 to 2000 m/z, nebulizer gas flow rate of 8 L/min, pressure of 10 psi, and fragmentor of 175 V. The mass correction was performed on the same way that for the targeted analysis. For MS/MS analysis, auto MS/MS acquisition mode was employed for the analysis of one of the samples employing a mass range between 100 to 3000 m/z, a scan rate of 6.0 scans/s, isolation width of 1.3 amu,