Electrochemical behavior of mitragynine and its determination by HPLC with dual-channel coulometric detection

This study provides the first report on the intrinsic electrochemical behavior of mitragynine, the principal alkaloid of Mitragyna speciosa (kratom). Voltammetric analysis revealed that mitragynine undergoes a pH-dependent anodic oxidation at relatively low potentials (Epa = 530 mV vs Ag/AgCl at pH 7), with a Nernstian slope of 59 mV per pH unit, suggesting a proton-coupled electron transfer mechanism involving the indole moiety. Under the studied experimental conditions, the mitragynine anodic feature is irreversible and diffusion-limited. Based on these electrochemical findings, a sensitive HPLC method with dual-channel coulometric detection (HPLC-ECD) was developed for the determination of mitragynine without requiring any prior derivatization steps. Under optimized conditions at +700 mV vs Pd/H₂, the method achieved a superior limit of detection (LOD) of 1.8 ng mL^-1 and a limit of quantification (LOQ) of 5.5 ng mL^-1, maintaining excellent linearity (R² = 0.999) across the concentration range of 20-200 ng mL^-1. The utility of the dual-channel configuration was demonstrated through the adaptive optimization of selectivity: while +700 mV ensured maximum sensitivity for kratom plant extracts (yielding 13.5 mg g^-1), a lower potential of +300 mV effectively suppressed co-eluting endogenous interfering species in complex human plasma matrices. The use of dual-channel detection enabled not only the selectivity optimization, but also confirmation of analyte identity based on the consistency of signal ratios at different detection potentials. The proposed approach represents a simple, cost-effective, and reliable strategy for the determination of mitragynine and highlights the potential of electrochemical detection for the analysis of electroactive alkaloids in complex matrices.