A Rapid and Ultra-Sensitive LC-MS Method for Quantification of IKKβ Inhibitor VIII and its Release from Biopolymers

Abstract

IKKβ Inhibitor VIII, synonymously ACHP, is a potent and selective small molecule inhibitor of IKKβ employed to study and target the NF-κB signaling pathway. Due to the ubiquitous nature of NF-κB signaling in physiological systems, localized controlled delivery of NF-κB target molecules such as ACHP enables spatial and temporal control over dosing and minimizes side effects associated with oral administration. However, there are many challenges in the detection and quantification of small molecules due to low concentrations required for biologically relevant dosing, complexity of sample matrix, and also the selectivity, specificity, and sensitivity of detection methods. This work highlights streamlined sample preparation and method development for the robust and sensitive detection of ACHP using ultrahigh performance liquid chromatography (UPLC) coupled with high-resolution mass spectrometry (HRMS). The method was applied to characterize ACHP-loaded biomaterial delivery systems and to quantify in vitro drug release. Extraction was accomplished by a simple protein precipitation method, achieving high extraction efficiency with no interference from the biomatrices. Good linearity (r > 0.99) was observed between concentrations 5 ng/mL to 2000 ng/mL. Intra- and Inter-assay variabilities were less than 5%, and the lower limit of detection (LOD) was 63.68 ± 6.29 ng/mL, which is within efficacy range. The developed UPLC-Q-ToF MS method was employed to quantify loading efficiencies and release kinetics of ACHP from biomaterials and demonstrated successful controlled release of the inhibitor in vitro.

Keywords: liquid chromatography, Mass spectrometry, UV-Vis Spectroscopy, Small molecule IKKβ Inhibitor, drug release, collagen