Integration of QRM and DoE in a QbD framework for developing a VEER chromatography method of IM-4E in rat plasma: Pharmacokinetic and greenness assessment.

Heterocyclic scaffolds with multifunctional therapeutic properties have gained significant attention in the field of drug discovery. Therefore, an environmentally sustainable analytical method based on ultrasound-assisted deep eutectic based miniaturized liquid-liquid extraction was developed to isolate the newly synthesized pyrazolyl flavone derivative, IM-4E, from rat plasma and to assess its pharmacokinetics. The RP-HPLC method was systematically optimized using an analytical quality by design framework integrating Ishikawa analysis, risk assessment, and design of experiments. Failure modes were identified and prioritized using failure mode and effect analysis based on the risk priority number. High-risk parameters were further investigated using fractional factorial and Box-Behnken designs to evaluate their effects on the tailing factor, theoretical plate number, and %recovery. Optimization through response surface methodology established a robust design space. Ultrasound-assisted extraction using a deep eutectic solvent composed of tetrabutylammonium chloride and 1-dodecanol (2:1) yielded the highest extraction efficiency. Chromatographic separation was achieved using a Thermo Fisher ODS analytical column (250 mm × 4.6 mm, 2.7 μm) with a mobile phase of 0.1% formic acid and methanol (30:70, v/v) and detection at 260 nm. The method demonstrated good linearity over the concentration range of 5.00-10,000.00 ng mL-1 (r = 0.9993), with limits of detection and quantification of 0.04 and 0.13 ng mL-1, respectively. Precision studies demonstrated acceptable reproducibility and repeatability of the results. The validated method was successfully applied to evaluate the bioavailability of IM-4E in male Wistar rats. The greenness of the method was analysed using AGREE, AGREEprep, MoGAPI, BAGI, and RAPI metrics.