Quantification by UPLC-MRM ESIMS of bufadienolides in Bryophyllum pinnatum leaves and manufactured products

Bryophyllum pinnatum is a succulent perennial plant native to Madagascar. It is used in anthroposophical medicine to treat psychiatric disorders, and as a tocolytic agent to prevent premature labour. Besides flavonoids, the plant is known to contain bufadienolides, which reportedly exert sedative and positive inotropic as well as central nervous system-related activities. Despite the possible toxicological relevance of bufadienolides, no reliable data are available about their content in plants and phytotherapeutic preparations. In this context, a UPLC- ESIMS assay with multiple reaction monitoring (MRM) has been developed and validated for the quantification of the main four bufadienolides in B. pinnatum, bersaldegenin-1-acetate, bryophyllin A, bersaldegenin-3-acetate, and bersaldegenin-1,3,5-orthoacetate. The separation was performed on a Kinetex 1.7 uXB-C18 column with a gradient of acetonitrile/water containing 10 mM ammonium formiate. Bufalin was used as an internal standard. Reference compounds were previously isolated from the related species B. daigremontianum. The method was used to determine the content of these four bufadienolides in B. pinnatum leaves of different geographical origins, and in manufactured products such as pressed juice and dried powder.