Combined green analytical principles and quality by design for ultraperformance liquid chromatography analytical method development, the characterization and in-silico toxicity prediction of Ixazomib degradation products using mass spectrometry

Abstract

Ixazomib citrate (IC) is the first oral selective proteosome inhibitor for treating multiple myeloma. IC is prone to degradation due to its oxidative deboronation and the amide bond, affecting patient health, drug quality, and efficacy. The stability of IC is crucial during drug development as it guides the inherent stability of the molecule, its degradation pathways, packing materials, and formulation development. Following the International Conference on Harmonization (ICH) Q1A (R2) and Q1B, a stability study were performed for both solution and solid-state stress studies. Under oxidative and alkaline conditions, 3 degradation products (DPs) were identified, separated, and method-validated according to ICH Q2 (R1) guidelines. From the Design Expert statistical tool, the Central Composite Design was used to optimize the final analytical method conditions, where the p-values for the model are < 0.05%. Green analytical chemistry has significantly reduced the use of hazardous organic solvents without losing chromatographic performance. The green separation and quantification of DPs and IC on Ultra Performance Liquid Chromatography (UPLC) using an Inert-Sustain C8 (50×3.0) mm 2.0 µm column with gradient elution using 10 mM ammonium acetate buffer (pH 5.0) and ethanol at a flow rate of 0.5 mL/min and detection at 230 nm. The results of green assessment tools like GAPI, AGREE, and Analytical eco-scale found that the method is excellent for the greenness of utilizing ethanol as a solvent, shorter runtime, and lesser waste. The method was validated as per ICH Q2 guidelines, and the results found it is sensitive, precise, accurate, robust, and linear for its intended use. The method is suitable for quantifying IC and its DPs from 2.0 to 150 µg/mL with R2 values of 0.9996 with a detection limit of 1.0 µg/mL. The plausible degradation structures and pathways of DPs were outlined using tandem mass spectra employed on LC-QTOF-MS/MS in both ESI positive and negative modes. The mechanistic explanation for establishing DPs was explained in detail. The ADMET Predictor™ software predicted the physicochemical and ADMET properties. The toxicity profile reveals that DP2 and DP3 are teratogenic, while D1 and D3 show phospholipidosis.