Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth.

Reports of therapy failures related to the use of daptomycin (DAP) are steadily increasing. This is mainly due to emerging DAP resistance for which, however, the underlying mechanism is often unknown. To elucidate the mode of action of novel DAP resistance traits it is indispensable to reliably detect and quantify DAP in complex matrices such as bacterial culture media. In this study, we established a selective and sensitive quantification method for DAP upon growth of a DAP resistant Mammaliicoccus sciuri strain in Mueller-Hinton medium. The method combined methanol-induced protein precipitation followed by high performance liquid chromatography with gradient elution coupled to mass spectrometry (LC-MS/MS) using daptomycin-d5 as internal standard. All validation parameters met the acceptance criteria of the European Medicines Agency (EMA) guideline on bioanalytical method validation. We successfully applied this highly selective and sensitive LC-MS/MS method for the quantification of DAP in in vitro studies addressing DAP resistance mechanisms in Gram-positive bacteria.