**3. Extraction Techniques**

Several methods for assaying these compounds have been studied, mainly for the purpose of monitoring certain drugs used in traditional Chinese medicine. The main issue concerns the difficulty of obtaining access to standard material for the setup and validation of analytical methods required for MBG measurement [68–72]. Generally, bufadienolides are extracted through solvent treatment from the dry or fresh skin and secretions of toads. More often, chlorinated and non-chlorinated organic solvents or alcohols are used and various techniques have been developed to separate MBG from other solutes, such as classic column chromatography, thin-layer chromatography, preparative-scale high performance liquid chromatography (HPLC) and flash column chromatography [68,73–78].

One of the first extraction techniques to obtain MBG was described by Shimada et al. in 1979. He retrieved this extract by soaking 40 *Bufo marinus* toad skins in ethanol and then dividing them into an ethyl–water acetate system. Later, the aqueous component was broken down via chromatography on Amberlite XAD-4 to split the conjugated steroids. The resulting fractions were purified via HPLC or gel chromatography on Sephadex LH-20 [79]. A different technique used by Bagrov et al. was able to separate MBG from the crystallized poison of Bufo marinus using thin layer chromatography. Today, the most widely used method for chemical characterization is mass spectrometry, allowing us to

differentiate these polar molecules based on their mass spectral fragmentation paths [28]. An ELISA enzyme test kit is available for the correct measurement of MBG. Enzymatic reactions can be then quantified with an automatic photometer for microplates [20].

MBG has been extracted from human plasma and urine [8,25,80–83], and there is the possibility of measuring MBG in 24h urine samples in the presence of other steroid hormones, through solid-phase dissociation-enhanced lanthanide fluorescent immunoassay, based on a 4G4 anti-MBG mouse monoclonal antibody [80].
