Development and Validation of a Multianalyte LC-MS/MS Method for Quantification of BRAF and MEK Inhibitors and Cannabidiol in Cell Lysates and Cell Culture Media

Background: BRAF and MEK inhibitors are established targeted therapies for melanoma and are frequently administered in combination regimens (dabrafenib/trametinib, encorafenib/binimetinib, and vemurafenib/cobimetinib). The non-psychoactive phytocannabinoid cannabidiol (CBD) is currently under investigation as a potential anticancer compound. As CBD may be combined with targeted anticancer therapies, potential effects on intracellular drug exposure require further investigation. Methods: A rapid and sensitive LC-MS/MS method was developed and validated with reference to the relevant principles of the ICH M10 guideline for the simultaneous quantification of three BRAF inhibitors, three MEK inhibitors, and CBD in cell lysates and cell culture media. The method was applied to determine intracellular drug levels in A375 melanoma cells following single-agent and combined treatments. Results: The method showed robust analytical performance across the evaluated validation parameters, including selectivity, calibration performance, precision, accuracy, recovery, and stability, and met the predefined acceptance criteria where applicable. Because of limited apparent analyte recovery in protein-free aqueous solutions, most likely resulting from nonspecific surface adsorption of the lipophilic compounds, 1% (w/v) bovine serum albumin was added for analyte stabilization. Application of the method to A375 melanoma cells enabled the reliable detection of treatment-dependent alterations in protein-normalized cell-associated analyte levels. Conclusions: This validated LC-MS/MS method enables reliable simultaneous quantification of BRAF inhibitors, MEK inhibitors, and CBD in cellular models. It provides a valuable tool for investigating intracellular drug concentrations and potential drug–drug interactions affecting intracellular drug levels.