Development and validation of a spectrophotometric method for the quantification of total bufadienolides in samples of toad glandular secretions.

Background: Bufadienolides are the main secondary metabolites found in the paratoid gland secretions (PGS) of toads of the Bufonidae family. These compounds are considered the main bioactive components of PGS. The aim of this study was to develop and validate the first method for the quantification of total bufadienolides (free and esterified) in samples of paratoid secretions from toads, using the UV-Vis absorption spectrophotometry technique.

Methods: The proposed method was based on the bathochromic shift induced by the reaction of the α-pyrone group of bufadienolides (296 nm) with a 5% (w:v) aqueous solution of sodium hydroxide and detection at 356 nm, after 60 min (time defined based on the evaluation of kinetic assays).

Results: The proposed method showed wide linearity (r = 0.9999), low LOD (1.3 × 10^-4 µg/mL) and LOQ (3.9 × 10^-4 µg/mL), recovery (84%-99%), repeatability (%RSD ≤ 5), reproducibility and robustness (p > 0.05). The total bufadienolide content in PGS extracts from 12 samples of R. diptycha ranged from 478 to 801 mg of EqMB/g of extract, while the R. granulosa sample presented 661 mg of EqMB/g of extract.

Conclusion: The new developed method is innovative, simple, fast, accurate, robust, low cost, and can contribute to future research focused on the quantification of total bufadienolides in samples of toad glandular secretions. In addition to serving as a strategic tool in the selection of work matrices, optimizing time, and minimizing costs.