From vial to injector: Stability, degradation, and surface interactions of non-derivatized ecgonine methyl ester in GC–MS

Ecgonine methyl ester (EME) is a primary metabolite of cocaine widely used in forensic toxicology, yet it is often regarded as problematic for direct GC–MS analysis due to presumed thermal instability and surface-related losses. In this study, the stability of EME and its isotopically labeled analogue (EME-d₃) was systematically investigated under realistic laboratory conditions relevant to forensic workflows. Injector temperature, solvent composition, storage conditions, container material, evaporation steps, and matrix effects were evaluated using GC–MS. The results demonstrate that EME does not undergo significant thermal degradation during GC–MS injection, even at elevated injector temperatures, and that in-injector formation of anhydroecgonine methyl ester (AEME) is negligible. Instead, AEME formation arises predominantly from slow, solution-phase dehydration prior to injection, strongly influenced by solvent type, pH, vial material, and matrix composition. Methanol promoted degradation, whereas acetonitrile and mild acidification markedly improved stability. Biological matrices, particularly urine extracts, exerted a protective effect, suppressing dehydration during autosampler storage. A pronounced positive matrix effect was observed for EME in GC–MS, particularly in urine extracts, and was only partially compensated by the isotopically labeled internal standard. Adsorptive losses during evaporation were a major preanalytical issue: nearly quantitative in polypropylene and only partially mitigated by a keeper in glass vials. Overall, these findings show that the analytical challenges associated with EME are primarily preanalytical rather than instrumental. When appropriate handling strategies are applied, EME and EME-d₃ can be reliably quantified by GC–MS without derivatization, supporting their broader use in forensic toxicology. • EME and EME-d₃ are thermally stable under routine GC–MS injection conditions, showing no injector-induced conversion to AEME up to 290 °C. • Analytical challenges associated with EME arise predominantly from preanalytical handling and surface interactions rather than from GC–MS instrumentation. • Methanol promotes slow, solution-phase dehydration of EME during storage, whereas acetonitrile provides significantly improved chemical stability. • Evaporation causes severe adsorptive losses of EME, particularly in polypropylene containers; keeper addition only partially mitigates losses in glass. • Positive matrix-induced signal enhancement of EME was demonstrated in GC–MS, challenging the assumption of negligible matrix effects under EI conditions.