Quantificação de bufadienolídeos nos venenos de Rhinella Marina e Rhaebo Guttatus por CLAE-UV

Abstract

Bufadienolides are the major active compounds of Bufonidae family animals. Recent studies have reported cytotoxic and antitumor activity of these molecules. A high performance liquid chromatography method with UV detection was developed and validated to quantify three bufadienolídeos (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of venoms of toads Rhinella marina and Rhaebo guttatus. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 x 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 recovery values (86% to 111%) adequate. The limits of detection of the bufadienolides were from 7.4 µg mL-1 for telocinobufagin, 4.2 µg mL-1 for marinobufagin and 4.0 µg mL-1 for bufalin. The intra-day and inter-day precisions of the method were evaluated and had a standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of Rhinella marina, which showed a content of 60% of marinobufagin. The same method was not selective for Rhaebo guttatus venom extract, although performing linear, accurate and precise, requiring the development of a technique that presents a higher selectivity.