Simultaneous Estimation of Avutometinib and Defactinib in Rat Plasma by Using LC–MS/MS and Its Application to Pharmacokinetic Studies

ABSTRACT The combination of avutometinib, a dual RAF/MEK inhibitor targeting the MAPK pathway, and defactinib, a selective focal adhesion kinase (FAK) inhibitor, represents a recently approved combination therapy for patients with KRAS‐mutated or KRIS‐mutated recurrent LGSOC (low‐grade serous ovarian cancers). A comprehensive review of the literature indicates that although clinical studies have demonstrated the therapeutic potential of this combination, limited information is available regarding simultaneous quantification in biological matrices. Therefore, the present study mainly focused on a rapid selective and specific LC–MS/MS method that was developed and fully validated for the estimation of avutometinib and defactinib in rat plasma. Chromatographic separation was achieved using a reverse phase Kinetex PS C18 2.1*50 mm, 3.5 um, 100A° column under gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phases. The flow rate was 0.6 mL/min, and glyburide was employed as the internal standard (IS). Mass spectrometric detection was performed using positive electrospray ionization mode and multiple reaction monitoring was applied for the quantitative analysis. Protein precipitation with acetonitrile was used for sample preparation. The method demonstrated an excellent linearity range of 1 ng/mL to 1000 ng/mL for both analytes ( r 2 > 0.99). Intraday and interday accuracy and precision were observed to be within the acceptable regulatory limits for all quality control levels. Considering the limited availability of validated bioanalytical methods for this drug combination, the present work provides a robust and sensitive LC–MS/MS approach for the simultaneous quantification of avutometinib and defactinib. This validated method was successfully applied to a rat pharmacokinetic study following oral administration of both analytes.