A Two-Stage In Silico-Guided Workflow for Forensic Toxicology: Empirical Validation via Capillary Zone Electrophoresis Prior to Mass-Spectrometric Confirmation

Medium-throughput forensic toxicology laboratories are increasingly expected to detect highly polar metabolites while working under tight resource and time constraints. To meet these requirements, a workflow is proposed that includes two stages: The first is computational metabolite prediction, followed by capillary zone electrophoresis (CZE), and the second stage is mass spectrometry (MS). The predictive step generates plausible metabolites and relevant physicochemical properties, which help guide early separation strategies. CZE then provides a rapid, low-cost way to test these predictions, identify informative samples, and exclude those unlikely to yield meaningful findings. Only samples that warrant further investigation proceed to targeted LC–MS/MS or high-resolution MS for confirmation. This approach shifts analytical effort toward the least resource-intensive stages, reducing unnecessary MS runs and improving turnaround time without compromising evidentiary standards. In practice, the workflow also improves day-to-day laboratory efficiency by overcoming equipment limitations and helping analysts focus on samples with genuine interpretive value. This stepwise combination of techniques is therefore suitable for routine forensic casework, where analytical decisions must be transparent, reproducible, and defensible.