Pulsed amperometric detection: Extending the applicability of HPLC to substances with two-step redox processes.

Abstract

Electrochemical detection coupled to liquid chromatography provides high selectivity compared to optical detection due to the lower number of electrochemically active compounds. Nevertheless, not all electrochemically active substances may be detected with the classical amperometric detection that involves the application of a single potential. For instance, in flow conditions, the main biomarkers of cinnamon (eugenol, coumarin, cinnamaldehyde and cinnamic acid) require an oxidation step prior to their amperometric detection. Pulsed amperometric detection (PAD) is proposed to address the electrochemical determination of substances with such complex redox behaviour in flow conditions because it allows the application of multiple potential steps that mimic the sequential stages that would be performed in static voltammetric measurements. PAD waveform has been optimized resulting in two scenarios (tox>> tred and tox <<tred) with different detection mechanisms involved that lead to either upward or downward peaks. In both scenarios, a notable analytical performance was achieved for all considered compounds and the proposed method was successfully validated for the analysis of a cinnamon sample, obtaining statistically comparable results to those obtained by HPLC-UV. The HPLC-PAD approach proposed in this work will extend the applicability of HPLC-ED to a wider set of substances that include not only those compounds that can be determined by a direct oxidation or reduction process but also those that require a two-step redox process. This will have a positive impact on targeted food analysis, in which it will facilitate the determination of electrochemical biomarkers in the presence of complex matrix samples.