Figure 1.

Total ion chromatograms of negative and positive ions of metabolites and peptides from Agkistrodon piscivorus leucostoma venom. The negative ion peak that dwarfs all others is citric acid. Assuming no metabolite loss during deproteination, the metabolites and peptides separated here represent the small molecule component of ~69 µg of crude venom. Metabolites were separated on a SeQuant ZIC-pHILIC HPLC 2.1 × 150 mm column, flow rate 120 µL/min, using acetonitrile as solvent A, and 10 mM ammonium carbonate, 0.1% ammonium hydroxide in water as solvent B. Separation was done in HILIC mode, with a linear gradient from 20% to 80% solvent B in 30 min, followed by a wash for 20 min with 20% acetonitrile, 0.5 M sodium chloride in water (solvent C) and, finally, column re-equilibration with starting conditions for 15 min.