BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF COMPOUND-9(O) USING LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY AND ITS APPLICATION TO A PHARMACOKINETIC STUDY

Abstract

Objectives: A sensitive, specific, robust, and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated in mouse plasma for a novel Secretory Phospholipase A2(SPLA2) Inhibitor, compound-9(O), and followed by its application to a Pharmacokinetic (PK) study. Methods: A sensitive and robust LC–MS/MS bioanalytical method was developed and validated for the quantification of compound-9(O) in biological matrices. Chromatographic separation was achieved using an isocratic mobile phase consisting of 0.1% formic acid in 2 mM ammonium formate prepared in typeI water and 0.1% formic acid in acetonitrile in the ratio of 20:80 (v/v). Separation was performed on a Kinetex® Biphenyl column (100×4.6 mm, 2.6 μm, 100 Å). Sample preparation was carried out using a protein precipitation technique with acetonitrile. Results: Intra- and inter-run accuracy ranged from 96.67% to 112.88% and 96.33% to 108.5%, respectively. The intra- and inter-day precision values were 1.46–6.95% and 0.96–8.54%, respectively. All stability assessments were within acceptance criteria. Conclusions: The validated LC-MS/MS bioanalytical method showed excellent specificity and selectivity, with satisfactory linearity over the validated concentration range from 1.00 to 1000 ng/mL. Accuracy, precision, ruggedness, and stability met the acceptance criteria as per ICH M10 guidelines, confirming the robustness of the method. Owing to its simplicity, reliability, and reproducibility, the validated method is well suited for routine analysis and can be effectively applied in PK, acute, subchronic, and chronic toxicokinetic studies.