Determination of cobimetinib in human plasma using protein precipitation extraction and high-performance liquid chromatography coupled to mass spectrometry

• A protein precipitation extraction LC-MS/MS assay was validated to analyze Cobimetinib in human plasma.
• The intra- and inter-assay precision and accuracy meet the FDA bioanalytical validation guideline.
• The assay was successfully used to support the Cobimetinib clinical trials and NDA filing.

Inhibition of MAP/ERK kinase (MEK) is a promising strategy to control the growth of tumors that are dependent on aberrant signaling in the MEK pathway. Cobimetinib (GDC-0973) (S)-[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone) inhibits proliferation of a variety of human tumor cell lines by inhibiting MEK1 and MEK2. A specific high performance liquid chromatography-mass spectrometric assay was developed and validated for the determination of cobimetinib in human plasma. The overall mean recovery using protein precipitation extraction with acetonitrile was found to be 54.1%. The calibration curve was ranged from 0.20 to 100 ng/mL. The LLOQ was sensitive enough to detect terminal phase concentrations of the drug. The intra- and inter-assay precision (%CV) was within 10.3% and 9.5% for cobimetinib. The assay accuracy (%RE) was within ±13.7% of the nominal concentration values for cobimetinib with the normal analytical QCs. The developed assay was successfully used to analyze the human plasma samples (for pharmacokinetic analysis) from clinical trials.