The use of Electrochemsiry is a complementary approach to traditional methods such as in vivo (human, rodent) or in vitro (liver microsomes) metabolism studies, and delivers the oxidative metabolic fingerprint of a (drug) molecule in a very short time. The acquired mass spectra are presented in simple 2 dimensional or more illustrative 3 dimensional plots, so-called MS voltammograms. A MS voltammogram visualizes the ion abundance versus m/z as a function of applied potential to the electrochemical cell. With a MS voltammogram the optimal potential can be determined for electrochemical generation of the desired metabolite for further research, e.g., a phase II metabolism study (i.e. adduct formation). It is a quick method to identify reactive pathways of the compound of interest. Additionally, electrochemistry allows to trace the reactive metabolite conjugates with targets (e.g., proteins, glutathione) without matrix interactions in contrary to traditional methods. A dedicated software program has been developed to automate and simplify the MS voltammogram acquisition. The program controls the syringe pump, the potentiostat and triggers the acquisition of mass spectra at the designated cell potentials. The total acquisition time needed for recording of a full MS voltammogram can be as short as 5 minutes. Amodiaquine an anti malaria agent was chosen as one of the model drugs to investigate oxidative metabolism using the on-line EC/MS system with automated MS voltammogram acquisition The easy and fast Electrochemical conversion of Amodiaquine into its 4 major phase I metabolites will be presented. In a second step Glutathione (GSH) is added to the electrochemically generated metabolites to form the appropriate GSH-metabolite adducts, mimicking phase II reactions. All known adducts were successfully formed and identified with MS. Additionally, MS voltammograms of other drugs and xenobiotics (e.g., acetaminophen, amiodarone, irinotecan) are presented. The data demonstrate that hyphenation of electrochemistry with electrospray mass spectrometry provides a versatile and user-friendly platform for rapid and cost efficient screening of target compounds (drugs, xenobiotics, etc.) in phase I and phase II metabolomics studies.

Recently, the scope of Electrochemistry (EC) upfront MS has been extended from mimicking drug metabolism towards new applications such as: protein/peptide cleavage, disulfide bonds reduction, covalent drug-protein binding, etc. In this presentation we will show the application of on-line EC/MS as a powerful tool to simulate various oxidation and reduction processes in life sciences. A specially designed μ-preparative electrochemical flow cell will be presented. The cell allows the synthesis of sufficient amounts of metabolites in a few minutes for subsequent use as reference material (e.g. NMR or MS). New scanning method was applied for oxidation of the highly concentrated samples (mM range) to achieve high yield in the metabolites formation. Stable oxidation conditions were obtained without the need of any cell maintenance for a prolonged period of time. Electrochemistry up front MS can be applied for protein and peptide cleavage (as a promising new approach to enzymatic digestion). Electrochemical cleavage of proteins and peptides occurs very specifically at C-terminal of the Tyrosine and Tryptophan peptide bonds. Examples of oxidative cleavage will be presented. Disulfide bonds are one of the most important post-translational modifications for proteins. In this poster we present the structural analysis of biologically active peptides and proteins containing disulfide bonds (e.g., somatostatin, insulin, etc) using electrochemistry (EC) combined with mass spectrometry. Therefore the sample undergoes electrolytic disulfide cleavage in the electrochemical flow cell followed by online MS analysis. Based on the intact protein mass and the resulting fragments and the MS/MS data unambiguous assignment of the disulfide bonds becomes possible. All these applications illustrate the tremendous power and broad applicability of electrochemistry as a tool to mimic nature's Redox reactions within a few seconds or minutes.

RP-65 Combining Electrochemistry (EC) with Mass Spectrometry (MS) has shown great potential for the investigation of drug metabolism1,2,3. Recently, the use of EC/MS has been extended towards new applications such as: 1. Fast synthesis of metabolites in micro preparative mode to generate sufficient amounts for the characterization by NMR and/or use as reference material. A specially designed μ-preparative electrochemical flow cell will be presented. 2. Rapid risk assessments of drug-protein binding. Investigation of drug-protein adducts by conventionally used techniques (microsomal incubation, in-vivo studies) are very laborious and have often low efficiency. With the application of EC, it is possible to activate proteins and drugs within seconds to undergo covalent drug-protein binding. 3. Signal enhancement in MS Proteomics. EC/LC/MS can enhance the signal intensity in MS by using electroactive derivatizing agent (e.g. N-(2-Ferroceneethyl)maleimide (FEM) for stabilizing thiol groups in proteins) or directly improving ionization efficiency. 4. Oxidative damage of DNA. On-line EC/ESI-MS is novel tool to study oxidative processes of nucleic acids, as well as to create covalent drug adducts with nucleic acids. All these applications illustrate the tremendous power and broad applicability of electrochemistry as a promising tool to mimic nature's Redox reactions, including oxidative damage of DNA, protein stress, lipid oxidation, etc. [1] Jurva U., Wikstrom H. V., Weidolf L., Bruins A.P., Comparison between electrochemistry/mass spectrometry and cytochrome P450 catalyzed oxidation reactions, Rapid Commun. Mass Spectrom. 17 (2003) 800–810. [2] Baumann A., Lohmann W., Schubert B., Oberacher H., Karst U., Metabolic studies of tetrazepam based on electrochemical simulation incomparison to in vivo and in vitro methods, J. Chromatogr. A 1216 (2009) 3192–3198. [3] Lohmann W., Hayen H., Karst U.,Covalent Protein Modification by Reactive Drug Metabolites Using Online Electrochemistry/LiquidChromatography/Mass Spectrometry, Anal. Chem. 80 (2008) 9714–9719.

RP-62 Reactive Oxygen Species (ROS) are a result of normal metabolism processes but in contact with toxic factor radiation or carcinogenic substance can cause nucleic acids modification and lead to DNA damage. Additionally, some drugs may form covalent bonds with nucleic acids resulting in such modifications. The DNA repair enzymes are monitoring the alterations, but unless errors are not fixed cell death, malignancy and aging may occur. Combining on-line electrochemistry (EC) with electrospray mass spectrometry (EC/ESI-MS) provides a powerful tool for simulating multiple oxidation processes, and among others oxidation of nucleic acids. ESI-MS allow directly monitoring of the reaction products and elucidating their structures by means of fragmentation experiment (MS/MS). In this poster we present the application of on-line EC/ESI-MS to study oxidative processes involving nucleic acids. It is demonstrated that EC is a useful tool for the activation of drug compounds to form covalent adducts with nucleic acids. EC/ESI-MS can be used for large-scale studies on the impact of sequence, structure, and modifications on nucleic acid oxidation as well as for assessing the risk of adduct formation between nucleic acids and chemicals. Moreover, on-line coupling of EC with liquid chromatography (LC) and MS, i.e., EC/LC/MS, is presented. Mass voltammograms containing valuable quantitative information about all major components were measured successfully. Ion suppression effects, which are usually observed in on-line EC/MS and which can severely hamper quantitative experiments, are excluded or at least reduced via chromatographic separation prior to mass spectrometric detection. In addition, on-line EC/LC/MS allowed identification of two different guanosine dimers after conjugation with acetaminophen (APAP).

RP-61 The knowledge of the metabolic pathways and the biotransformation of new drugs are crucial for elucidation of degradation routes of new active compounds, especially in the area of possible toxicity. In vitro studies are based on incubating drug candidates with e.g., liver cells (in microsomes activity of cytochrome P450 is high) and isolating and detecting the metabolic products. With the introduction of an on-line electrochemistry/mass spectrometry system (EC/MS), oxidative metabolism, as usually occurring in the liver cells by the Cytochrome P450 oxidation, can be simulated successfully within seconds and detected by electrospray mass spectrometry. Combining electrochemistry with MS creates a robust platform for oxidative metabolite investigations and helps to overcome many of the laborious tasks by isolating the metabolites form in vivo (urine, plasma, etc.) or in vitro (microsomes) studies. Acetaminophen (paracetamol; APAP; IUPAC: N-(4-hydroxyphenyl) acetamide) was chosen as model drug to investigate oxidative metabolism using the on-line EC/MS System dedicated for single component screening. Electrochemical conversion of the acetaminophen into reactive phase I metabolite (N-acetyl-p-benzoquinoneimine, NAPQI) and the NAPQI – glutathione (GSH) phaseII conjugate was successfully achieved. The data can be presented as 3-D mass voltamogramm, which represents ion abundance versus m/z as a function of EC potential.Mass voltammogram can be recorded automatically to obtain a metabolic fingerprint of the compound of interest in a short time frame. Hyphenation of on-line electrochemistry and electrospray mass spectrometry provides a versatile and user-friendly platform for screening of single target compounds (drugs, xenobiotics, etc.) in phase I and II metabolomics studies.