Development and Validation of a High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) Method for the Simultaneous Quantification of Ibrutinib and Diazepam in Human Plasma: Application in Pharmacokinetic Studies

Abstract

This study presents the development and validation of a novel analytical method for the simultaneous quantification of Ibrutinib (IBR) and Diazepam (DIA) in human plasma using Reverse Phase High Performance Liquid Chromatography coupled with Mass Spectrometry (RP-HPLC-MS/MS). The method is based on liquid-liquid extraction (LLE) followed by chromatographic separation on an Agilent Zorbax SB-C18 column and detection using a quadrupole mass spectrometer in positive ion mode. The method was carefully optimized to ensure high sensitivity, specificity, and reproducibility across a broad concentration range. Analytical validation was performed following the ICH M10 guidelines, assessing parameters including specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). The calibration curve demonstrated excellent linearity in the range of 0.5–40 ppb for Ibrutinib and 0.5–40 ppb for Diazepam. The method exhibited low inter- and intra-day variability with coefficients of variation (CV) below 10%. Matrix effects and the potential for interference were minimized, confirming the robustness of the method. Recovery rates for both compounds ranged from 90% to 105%. The developed method was successfully applied to the analysis of plasma samples in pharmacokinetic studies, offering a reliable and sensitive approach for the simultaneous quantification of Ibrutinib and Diazepam in clinical settings. This method is suitable for routine therapeutic drug monitoring, pharmacokinetic studies, and clinical pharmacology research.