Different experimental conditions were necessary for the separations as follow:
The Ecuadorian sample (remaining half of the dried secretion) was dissolved in 1.2 mL of solution A (99.95% H2O, 0.05% trifluoroacetic acid) and clarified by centrifugation. 1 mL supernatant was subjected to reverse phase HPLC employing a Waters binary pump HPLC system fitted with an analytical column: Phenomenex Jupiter C-18 (4.6 × 250 mm). Peptides were eluted with a gradient formed from 100% solution A (99.95% H2O, 0.05% trifluoroacetic acid) to 100% solution B (80.00% acetonitrile, 19.95% H2O, 0.05% trifluoroacetic acid) for 240 min at a flow rate of 1 mL/min. Fractions of 1 mL were collected every minute. Detection at 214 and 280 nm was performed continuously.
The Costa Rican sample (0.5 mL of 3 mL) from 1999 was injected onto an HPLC system fitted with a semi-preparative C-18 Phenomenex Jupiter Column employing a gradient of 0–80% solution B for 80 min at a flow rate of 2 mL/min. Fractions were collected every minute. This analysis was performed on 08/03/1999.
The Ecuadorian sample (remaining half of the dried secretion) was dissolved in 1.2 mL of solution A (99.95% H2O, 0.05% trifluoroacetic acid) and clarified by centrifugation. 1 mL supernatant was subjected to reverse phase HPLC employing a Waters binary pump HPLC system fitted with an analytical column: Phenomenex Jupiter C-18 (4.6 × 250 mm). Peptides were eluted with a gradient formed from 100% solution A (99.95% H2O, 0.05% trifluoroacetic acid) to 100% solution B (80.00% acetonitrile, 19.95% H2O, 0.05% trifluoroacetic acid) for 240 min at a flow rate of 1 mL/min. Fractions of 1 mL were collected every minute. Detection at 214 and 280 nm was performed continuously.
The Costa Rican sample (0.5 mL of 3 mL) from 1999 was injected onto an HPLC system fitted with a semi-preparative C-18 Phenomenex Jupiter Column employing a gradient of 0–80% solution B for 80 min at a flow rate of 2 mL/min. Fractions were collected every minute. This analysis was performed on 08/03/1999.