A Thin-Layer Electrochemical Flow Cell Coupled On-Line with Electrospray-Mass Spectrometry for the Study of Biological Redox Reactions

Abstract

A three electrode thin-layer,¯ow-by electrode cell was coupled on-line with electrospray-mass spectrometry (i.e., ECaES-MS) for the study of biological redox reactions. The cell made use of a commercially available 6.0 mm diameter, offset glassy carbon disk working electrode, a AgaAgCl reference electrode, and a workingacounter electrode spacing gasket (16 mm), along with a home-built PEEK counter electrode block with a Pt foil (7.0 mm wide) counter electrode (cell volume ca. 1.1 mL). Off-line hydrodynamic voltammetry experiments and electrolytic conversion ef®ciency measurements using chronocoulometry were used to characterize the performance of the cell. On-line ECaES-MS conversion ef®ciency was found consistent with the off-line results and the ECaES-MS response time was measured as 5.1 s at a¯ow rate of 30 mLamin and 2.4 s at 62 mLamin. The use of this hybrid system for the study of the products of biologically relevant redox reactions was demonstrated using the oxidation of dopamine in aqueous CH 3 OH (pH 4.0) as a test case. Tandem mass spectrometry experiments provided evidence to conclude that the structure of the two major dopamine oxidation products observed in the ES mass spectra were 5,6-dihydroxyindoline and 5,6-hydroxyindole. Reaction of oxidized dopamine with the surrogate biogenic nucleophile benzene thiol resulted in the formation of mono-, di-, and tri-benzene thiol addition products. These spectra also indicated that the reaction of oxidized dopamine with benzene thiol was signi®cantly faster than the intermolecular cyclization reaction of this intermediate, which normally produces 5,6-dihydroxyindoline.

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

“…Since then, Van Berkel's group and others have comprehensively investigated the theoretical aspects of electrochemistry in electrospray [31][32][33][34][35][36][37][38][39][40][41][42] as well as made significant advances in electrochemical flow cell hyphenation with electrospray mass spectrometry (ESI-MS) [37,[43][44][45][46], and electrochemical derivatization strategies for analyte detection by ESI-MS [47][48][49][50][51]. Moreover, Bruins and coworkers have used EC-ESI-MS to study or mimic biologically relevant electrochemical reactions [52], such as phase I oxidative reactions in drug metabolism [53] or cytochrome P450 catalyzed reactions [54].…”

Metabolomic applications of electrochemistry/Mass spectrometry

“…These results demonstrate that this technique is capable of very closely simulating the proposed biotransformation reactions related to estrogen-dependent carcinogenesis [44]. It must be recognized that, when compared with enzymatic oxidation (e.g., cytochrome P450), EC oxidation leads to the formation of similar products for those enzymatic catalyzed reactions that are supposed to proceed through a mechanism initiated by a one-electron transfer oxidation [36,37]. Reactions initiated by hydrogen atom abstraction such as hydroxylation of aromatic rings without electron donating groups are generally not mimicked by EC oxidation.…”

“…The use of upstream decoupled EC flow cells in series with MS has been previously described as a means of synthesizing and characterizing likely drug metabolites [36,37,42]. Our studies have also included the synthesis of endogenous compounds using endogenous metabolite precursors [43].…”

“…Of particular relevance to metabolomics is the use of on-line EC reactions to produce likely metabolites using known metabolites as reactants [36,37]. Redox reactions conducted in combination with analytical and preparative scale separations and qualitative techniques such as MS or NMR may be a useful route to isolation of sufficient quantities for structural elucidation and confirmation.…”

Electroanalytical Flow Measurements–Recent Advances

“…A commercially available electrochemical flow cell was used in biological application with electrospray MS as reactor before the HPLC column to study tryptophan metabolites [114]. A thin-layer cell with electrospray MS was employed for the study of the products and intermediates of dopamine oxidation [115], and also the same cell was used for electrolytic-based preconcentration and matrix elimination experiments on-line with inductively coupled plasma MS [116].…”