Quantification and clinical validation of the selective MET kinase inhibitor DO-2 and its metabolites DO-5 and M3 in human plasma

DO -2 is a highly selective MNNG HOS transforming (MET) inhibitor. This deuterated drug is thought to diminish the formation of the Aldehyde Oxidase 1 inactive metabolite M3. For various reasons, quantification of DO -2 and its metabolites M3 and DO -5 is highly relevant. In this study, we present an ultra -performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify DO -2, M3 and DO -5. Rolipram served as the internal standard. Aliquots of 25 mu L were mixed with 100 mu L internal standard consisting of 10 ng/mL rolipram in acetonitrile. Separation of the analytes was achieved on an Acquity UPLC (R) HSS T3 column, utilizing gradient elution with water/formic acid and acetonitrile/formic acid at a flow -rate of 0.400 mL/min. Calibration curves were linear in the range of 1.00 - 1000 ng/mL for DO -2 and DO -5, and 2.00 - 2000 ng/mL for M3 in human plasma. The within -run and between -run precisions of DO -2, DO -5 and M3, also at the level of the LLQ, were within 12.1%, while the accuracy ranged from 89.5 to 108.7%. All values for accuracy, within -run and between -run precisions met the criteria set by the Food and Drug Administration. The method was effectively employed in the analysis of samples obtained from a clinical trial.