Ultra-fast eco-friendly UHPLC–MS/MS methodology for the quantification of ASP3026 in human liver microsomes: Evaluation of metabolic stability via in silico software and in vitro metabolic incubation

ASP3026 is a recently formulated and highly selective inhibitor designed to target the ALK kinase. ASP3026 efficiently inhibited ALK kinase activity and demonstrated superior selectivity at a panel of Tyr-kinases compared to crizotinib. The target of this investigation was to establish a highly accurate, fast, green, and highly sensitive Ultra-high performance liquid chromatography- Tandem mass spectrometry (UHPLC-MS/MS) technique for assessing the concentration of ASP3026 in human liver microsomes (HLMs). In vitro incubation, the metabolic stability of ASP3026 in HLMs was evaluated using this known approach. The validation steps for the UHPLC-MS/MS analytical technique in the HLMs were performed along with the bio-analytical method validation guidelines settled by the US-FDA. To increase the ecological sustainability of the current UHPLC-MS/MS system, a lower flow rate of 0.3 mL min⁻¹, a shorter elution duration of 1 min, and a reduced consumption of ACN have been implemented. A screening of the chemical structure of ASP3026 for hazardous alerts and metabolic lability was performed by the StarDrop package, that includes the DEREK and P450 modules. The analytical separation of ASP3026 and fenebrutinib (FNB) on the reversed phase Eclipse Plus C18 column was performed using an isocratic mobile phase approach. The calibration curve produced by the ASP3026 showed a linear association over the level range of 1–3,000 ng mL⁻¹. A study was conducted to evaluate the precision and accuracy of UHPLC-MS/MS technology in evaluating both intra-day and inter-day variations. The accuracy exhibited a range of −1.56%–7.33% across various days, and a range of −0.78%–10.66% within the same day. The ASP3026 underwent in vitro half-life and intrinsic clearance measurements, yielding values of 14.32 min and 56.62 mL min⁻¹ kg⁻¹, correspondingly. According to in silico software research, using minor modifications to the piperazine component or substituting the group in drug design has the potential to improve the metabolic safety and stability of novel derivatives in comparison to ASP3026.