The combination of electrochemistry and spectroscopy, known as spectroelectrochemistry (SEC), is an already established approach. By combining these two techniques, the relevance of the data obtained is greater than what it would be when using them independently. A number of review papers have been published on this subject, mostly written for experts in the field and focused on recent advances. In this review, written for both the novice in the field and the more experienced reader, the focus is not on the past but on the future. The scope is narrowed down to four techniques the authors claim to have the most potential for the future, namely: infrared spectroelectrochemistry (IR-SEC), Raman spectroelectrochemistry (Raman-SEC), nuclear magnetic resonance spectroelectrochemistry (NMR-SEC) and, perhaps slightly more controversial but certainly promising, electrochemistry mass-spectrometry (EC-MS).

Over the last 3 decades, electrochemistry (EC) has been successfully applied in phase I and phase II metabolism simulation studies. The electrochemically generated phase I metabolite-like oxidation products can react with selected reagents to form phase II conjugates. During conjugate formation, the generation of isomeric compounds is possible. Such isomeric conjugates are often separated by high-performance liquid chromatography (HPLC). Here, we demonstrate a powerful approach that combines EC with ion mobility spectrometry to separate possible isomeric conjugates. In detail, we present the hyphenation of a microfluidic electrochemical chip with an integrated mixer coupled online to trapped ion mobility spectrometry (TIMS) and time-of-flight high-resolution mass spectrometry (ToF-HRMS), briefly chipEC-TIMS-ToF-HRMS. This novel method achieves results in several minutes, which is much faster than traditional separation approaches like HPLC, and was applied to the drug paracetamol and the controversial feed preservative ethoxyquin. The analytes were oxidized in situ in the electrochemical microfluidic chip under formation of reactive intermediates and mixed with different thiol-containing reagents to form conjugates. These were analyzed by TIMS-ToF-HRMS to identify possible isomers. It was observed that the oxidation products of both paracetamol and ethoxyquin form two isomeric conjugates, which are characterized by different ion mobilities, with each reagent. Therefore, using this hyphenated technique, it is possible to not only form reactive oxidation products and their conjugates in situ but also separate and detect these isomeric conjugates within only a few minutes.

This thesis is part of the "Nanopatterned Electrode Surfaces for Proteomics, Drug Screening and Synthesis" project led by the principal investigator Mathieu Odijk from the University of Twente (UT) in close collaboration with Hjalmar Permentier and Rainer Bischoff from the University of Groningen (RUG). It is funded by the Dutch technology foundation "Stichting voor de Technische Wetenschappen" (STW). In 2017, the STW was reorganised to become "Toegepaste en Technische Wetenschappen" (TTW), one of the three research domains of the Dutch national research council "Organisatie voor Wetenschappelijk Onderzoek" (NWO). The research project follows up on a previous one that resulted in several publications combining microfluidic electrochemical (EC) methods with liquid chromatography (LC) and mass spectrometry (MS). 1-3 In its current iteration, the project collaborates with the University of Groningen (RuG) and the two investigators Rainer Bischoff and Hjalmar Permentier. Additional collaborators are Albert van den Berg (UT), Wouter Olthuis (UT), Sonia García Blanco (UT) as well as Uwe Karst (University of Münster) and the companies Antec Scientific, Lionix International B.V., and Bronkhorst High-Tech B.V. The project aims to develop new nano-patterned surfaces with integrated waveguides in order to integrate spectroscopic methods into the previously mentioned microfluidic EC-LC-MS methods. It envisions devices capable of surface-enhanced infrared absorption spectroscopy (SEIRAS) and/or surface-enhanced Raman spectroscopy (SERS), in order to observe the reactions taking place inside the microreactor, possibly in real-time. The desired applications of these methods are the synthesis of added value fine chemicals, drug metabolism mimicry and protein analysis, all on the microfluidic, integrated scale. The last two applications are part of the research work presented in this thesis. Thesis ambitionIn the following, the applications drug metabolism mimicry and protein analysis are briefly explained, their problems stated, and solutions proposed. Lastly, research goals for this thesis are derived from the proposed solutions. The two main ones for total protein analysis are top-or middle-down 20,21 and bottom-up / shotgun proteomics 22,23 , as shown in Figure 1.2, reused from GREGORICH ET AL. 19 The abbreviations MS/MS, protein ID and PTM refer to tandem mass spectrometry, protein identification and post-translational modification, respectively. Both approaches include sample processing steps such as separation via liquid chromatography (LC) and In this chapter, co-authored by Jasper Lozeman and Pascal Führer, an overview of recent advances in a selection of spectroelectrochemical (SEC) methods is given and critically discussed before giving recommendations for advancements. The selection comprises the combinations of electrochemistry with infrared and Raman spectroscopy, written by Jasper Lozeman, and nuclear magnetic resonance (NMR) spectroscopy as well as mass spectrometry, written by Pascal Führer.