Quantification of bufadienolides in the poisons of Rhinella marina and Rhaebo guttatus by HPLC-UV

Abstract

Bufadienolides are the main active compounds in the Bufonidae family of frogs. Recent studies have demonstrated cytotoxic and/or antitumor activity in these molecules. A HPLC-DAD method was developed and validated to quantify three bufadienolides (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of the cane toad poison frogs and smooth-sided toad. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 × 4.6 mm, 5 μm), using gradient elution with acetonitrile and water, at a flow rate of 1.0 mL min(-1) and detection at 296 nm. The method showed linearity (r > 0.999) and adequate recovery values (86%-111%). The limits of quantification of bufadienolides were 7.4 μg mL(-1) for telocinobufagin, 4.2 μg mL(-1) for marinobufagin and 4.0 μg mL(-1) for bufalin. Intraday and interday values of the method were evaluated and presented standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of the cane toad, which showed a content of 60% of marinobufagin. The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise, requiring the development of a technique that presents a greater selectivity.

Keywords: Bufadienolides; Method validation; Rhaebo guttatus; Rhinella marina.