Simultaneous Bioanalytical Method Development and Validation of Navitoclax and Doxorubicin in Rat Plasma Using UHPLC-HESI-LTQ-MS

Abstract

A simple, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was successfully developed and validated to determine navitoclax and doxorubicin in rat plasma. Ketoconazole and daunorubicin were employed as internal standards to ensure accurate quantification and method consistency. The sample preparation involved a straightforward protein precipitation technique, which facilitated efficient extraction of the analytes from the plasma matrix. The resulting supernatant was subjected to drying using a vacuum concentrator and later reconstituted before being injected into the LC-MS system. Separation was carried out using a SHIMADZU Shim-pack GIST C-18 column with mobile phase, consisting of a carefully balanced mixture of acetonitrile and water with 0.1% formic acid, with a flow rate of 0.5 mL/min. This composition ensured efficient elution and minimized matrix effects, contributing to the method's robustness and reproducibility. The developed method was comprehensively validated according to regulatory guidelines, assessing various parameters including specificity, selectivity, sensitivity, calibration curve performance, precision, accuracy, stability and dilution integrity. This bioanalytical method has the potential to be extended to various clinical settings, enabling the monitoring drug metabolism, monitoring potential drug-drug interactions and understanding the pharmacokinetic profile and adverse effect potential of the combination therapy.