Agilent 1260 infinity 2 high performance liquid chromatography

Lyophilized venom samples were hydrated in trifluoroacetic acid/distilled H₂O (dH₂O) 0.1/99.9 (v/v) to a final concentration of 0.0023 g/mL. Aliquots of 200 µL were injected into an InfinityLab Poroshell 120 Reverse Phase column (3.0 × 150 mm, 2.7 µm) and fractioned using an Agilent 1260 Infinity II High Performance Liquid Chromatography (HPLC) system (Agilent, Wilmington, DE, USA). Mobile phases were prepared as follows: mobile phase A, 0.1% trifluoroacetic (v/v) acid in double-distilled, sterile water; mobile phase B, LC-MS-grade acetonitrile. The separation was performed at a constant flow rate of 400 µL/min according to the following linear gradient of mobile phase B: 2% for 4 min; 12% at minute 6; 19.8% at minute 9; 39.2% by minute 34; 56% by minute 40; a ramp to 90% by minute 46; 1 min of wash; decrease to 2% in 2 min (at minute 49); and full re-equilibration until minute 53. The fractions were collected according to the following time scheme and were kept at 4 °C: minutes 0–12, no fractions collected; minutes 12–24, one fraction collected every 1.5 min; minutes 24–26, 1 fraction collected; minutes 26–38, one fraction collected every 1.5 min; minutes 38–53, no fractions collected. Venom elution was monitored using three wavelengths (214 nm, 260 nm, and 280 nm). Fractions were lyophilized and stored at −20 °C.