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

• A method by HPLC-UV was validated to quantify three bufadienolides in toad venom.
• The venom from Rhinella marina presented high percentage of marinobufagin (60%).
• The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise.

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.

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