Electrochemistry upfront MS Panacea: an ancient Greek goddess of healing. an answer or solution for all problems or difficulties. 1

ROXY™ - EC/LC upfront MS With ReactorCell™ for Generating Redox metabolites Cleavage of proteins/peptides Signal enhancement in MS Oxidative stress Metabolism 2

Why Electrochemistry / MS? Mimics Natures Redox Reactions In Drug Development and Proteomics Fast & Clean Complementary to existing methods

Why Electrochemistry / MS? Mimics Natures Redox Reactions Drug development Immediate results, minutes vs weeks No by products, no cleanup No biohazard No rodents Proteomics No enzymes, clean without by products

Electrochemistry / MS Mimicking nature’s redox reactions Fast alternative for laborious, time consuming procedures

Electrochemistry / MS Mimicking nature’s redox reactions Fast alternative for laborious, time consuming procedures EC MS

Modes of operation Off-line EC/MS On-line EC/MS On-line EC/LC/MS Pre injection - Post column

Electrochemical Reaction Oxidation of a dihydroxyphenyl derivate to 1,2 benzoquinone derivate, by dehydration

Role of electrode potential E Facilitates the EC reaction 20 40 60 80 100 400 800 1200 1600 E (mV) Signal (%) formation of oxidation or reduction products no reaction A  B

Role of electrode potential E Facilitates the EC reaction formation of secondary products 20 40 60 80 100 400 800 1200 1600 E (mV) Signal (%) 2000 B C A  B  C B: instable intermediate

oxidative degradation of the substrate How to Find Optimum ΔE ? Traditional Electrochemistry: Voltammogram On-line EC/ESI-MS: “MS Voltammogram” 180 200 220 240 260 280 300 320 340 360 380 400 1000 2000 3000 4000 5000 1500 2500 intensity [-] m/z E [mV] oxidative degradation of the substrate oxidation products

Typical Reactions Catalyzed by Cytochrome P450 Simulated by EC Allylic and aliphatic hydroxylation Possible at high potentials (only with ROXY) Benzylic hydroxylation Possible Desalkylation of amines Desalkylation of ethers and thioethers Ethers: Possible Tioethers: not mimicked Hydroxylation of aromatics Epoxide formation Oxidation of heteroatoms (N, S) R-CH2-CH3 R-CH-CH3 OH N-CH2-R NH + R-CHO R-O-CH2-R R-OH + RCHO O C=C R C C HO R C NH R R C+ N R O- H Adapted from W. Lohmann et al. LC/GC Jan. 2010 12

Proteomics Electrochemical cleavage (oxidation) of proteins/peptides ‘enzymatic digestion’ by electro-chemical cell no enzymes, no non-specific cleavage, no auto-digestion, etc. 13

Electrochemistry in Proteomics

Electrochemistry in Proteomics

Electrochemistry in Proteomics

Conclusion EC/MS Many application areas Time and cost savings... Fast and clean

Conclusion EC/MS Mimics Natures Redox Reactions Drug development Immediate results, minutes vs weeks No by products, no cleanup No biohazard No rodents Proteomics No enzymes, clean without by products

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