1. Mimicking Drug Metabolism using ROXY EC System
ASMS 2018
San Diego, CA, USA

2. Outline Introduction Drug metabolism – what you should know
Case Study 1 – Acetaminophen
Case Study 2 – Amodiaquine
Examples from Industry
Conclusions
The lecture will explore on-line EC/MS as a powerful technique to investigate various oxidation and reduction processes in life sciences.

3. Drug/Xenobiotic Metabolism
Many drugs metabolize in the liver by Cytochrome P450 enzymes
Drug metabolism consist of two phases:
Phase 1: oxidation, reduction, hydrolysis
Phase 2: conjugation (adduct formation)

4. Techniques used in Drug Metabolism
1. Test animals (rodents)  in-vivo
2. Microsomes  in-vitro
Rat Liver Microsomes (RLM) Human Liver Microsomes (HLM)
3. Electrochemical mimicking (= simulation)  in-electro

5. 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
Possible (?)
Oxidation of heteroatoms (N, S)
R-CH2-CH 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 C+ N R O- H
W. Lohmann et al. LC/GC Jan. 2010 5

6. Case Study 1: Acetaminophen
Paracetamol
Panadol
Tylenol
APAP

7. Metabolism of Acetaminophen
Phase 1
Phase 2

8. Instrumental Set-up Phase 1
Oxidative Metabolism (OxMet)
NAPQI

9. Positive vs. Negative ESI-MS of NAPQI
positive ESI
D. Tsikas, J.of Chrom. B, 879 (2011)

10. MS Voltammogram Acetaminophen
800 mV used for oxidation

11. Instrumental Set-up Phase 2
Conjugation with Glutathione (GSH)
NAPQI-GSH
NAPQI

12. Conjugation (Adduct Formation)
0 mV
NAPQI-GSH
NAPQI
800 mV

13. Zoom in: Mass Range
0 mV
800 mV

14. Case Study 2: Amodiaquine
Camoquin
Flavoquine

15. Metabolism of Amodiaquine

16. Mimicking Amodiaquine Metabolism by EC/MS
Cell OFF
400mV
1200mV
Metabolites 2 & 3
Metabolite 1
Amodiaquine

17. MS Voltammogram Amodiaquine
Met1
Met2
Met3

18. Conjugation with GSH
Met1-GSH
Met2-GSH

19. Examples form Industry

20. Biotransformation Pathway of Verapamil
LC/MS after EC oxidation and via incubation of HLM and RLM

21. Biotransformation Pathway of Verapamil
Molecular formula of Verapamil and it metabolites after EC oxidation and incubation with HLM and RLM

22. Biotransformation Pathway of Verapamil
Conclusions
Structure of electrochemically generated metabolites were elucidated
on basis of accurate mass data and MS/MS experiments
All of the metabolic products of Verapamil incl. Norverapamil can be
easily simulated using this purely instrumental technique
Furthermore newly reported metabolic reactions products like
carbinolamines or imine methides become accessible
EC/LC/MS is a powerful tool in drug discovery and development
providing faster and often more comprehensive results than in vivo
approaches
S. Jahn et al., J. of Chromatography A, 1218 (2011)

23. Generation of Statin Drug Metabolites
Simvastatin & Lovastatin (blockbusters)

24. less than 1 mg of drug = three oxidative metabolites of each parent
simvastatin
less than 1 mg of drug = three oxidative metabolites of each parent
complete NMR characterization was performed. For 3’α hydroxy SV complete 1HNMR first time ever!
the electrochemical approach gave higher yields than the enzymatic oxidations
lovastatin
Bluckbusters > 1 Billion, enzymatic variants
Anal. Bioanal. Chem. (2013) 405:

25. ThP141, 10:30 am - 2:30 pm

26. > 18 Publications using ROXY™ EC to
Mimic Drug Metabolism (last 15 months) 
Mechanism of aromatic hydroxylation of lidocaine at a Pt electrode under acidic conditions
T Gul, R Bischoff, HP Permentier - Electrochimica Acta, Elsevier
… 2.2. Electrochemical measurements. Constant potential measurements were performed with an Antec ROXY potentiostat (Antec Leyden, Zoeterwoude, The Netherlands) controlled by Antec Dialogue software. Cyclic voltammetry …
Electrochemical simulation of biotransformation reactions of citrinin and dihydroergocristine compared to UV irradiation and Fenton-like reaction
J Keller, H Haase, M Koch - Analytical and bioanal. chem, Springer
… μm spacers. An electrochemical potentiostat ROXY (Antec Leyden) was used to apply a potential ramped between … Electrochemical oxidation of phosphatidylethanolamines studied by mass spectrometry
S Colombo, G Coliva, A Kraj, J. of Mass …, Wiley Online Library
… The EC reactions were conducted in the ROXY EC system (Antec Scientific, Zoeterwoude, NL) consisting of the ROXY potentiostat for the control of the flow-through μ-PrepCell 2.0 …
Structural characterization of electrochemically and in vivo generated potential metabolites of selected cardiovascular drugs by EC-UHPLC/ESI-MS using an …
M Szultka-Młyńska, S Bajkacz, I Baranowska… - Talanta, Elsevier
… containing an electrochemical cell ReactorCell™, infusion pump and all the necessary HPLC connections (ROXY™ system Antec Leyden,..
Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos
TF Mekonnen, U Panne, M Koch - Analytical & bioanal. chem, Springer
… Mass voltammograms were generated by Roxy ™ potentiostat (Antec Leyden, Zoeterwoude, The Netherlands) equipped with μPrepCell ™….
Prediction of biotransformation products of the fungicide fluopyram by electrochemistry coupled online to liquid chromatography-mass spectrometry and comparison …
TF Mekonnen, U Panne, M Koch - Analytical and bioanalytical chemistry, Springer
… Mass voltammogram of FLP was recorded (continuous scan, 10 mV/s) using a Roxy™ potentiostat (Antec Leyden, Zoeterwoude) ...
Investigations of the Copper‐Catalyzed Oxidative Cross‐Coupling of Tetrahydroisoquinolines with Diethylzinc by a Combination of Mass Spectrometric and …
JA Willms, H Gleich, M Schrempp;  A European Journal, Wiley … Voltage/ion intensity curves were obtained with a commercial electrochemical flow cell [Antec Scientific ROXY potentiostat and …
Hydroxylation and dimerization of zearalenone: comparison of chemical, enzymatic and electrochemical oxidation methods
J Keller, H Haase, M Koch - World Mycotoxin Journal, 2017
… Oxidation of zearalenone by EC/MS, Fenton-like reaction and Ce(IV) was pumped through the µPrepCell by using a SP2-ROXY dual piston syringe pump (Antec Leyden) and a flow rate of 80 µl/min …
Laccase-and electrochemically mediated conversion of triclosan: Metabolite formation and influence on antibacterial activity
E Jahangiri, B Seiwert, T Reemtsma, D Schlosser - Chemosphere, 2017 – Elsevier … 2.4. Electrochemical (EC) conversion of TCS. TCS conversion was performed on a ROXY Potentiostat electrochemical system (Antec,.. 
Oxidation of 4-Isopropylphenol in Aqueous Solutions as a Model for Oxidation of Flavonoids to Form Dimers IG Zenkevich, TI Pushkareva - Chemistry of Natural Comp., 2018 Springer … Electrochemical oxidation of 4-isopropylphenol (1) (c 50–100 Pg/mL) was conducted in a mixture of aqueous NH 4 OAc (pH ~7.4) and MeCN (3:1) in a ROXY electrochemical cell (Antec, Leyden, …
Untargeted screening of phase I metabolism of combretastatin A4 by multi-tool analysis K Jaroch, PZ Goryńska, K Goryński, T Stefański… - Talanta, 2018 – Elsevier … ROXY™ system. All electrochemical experiments mimicking metabolism reactions in liver microsomes were conducted via the ROXY™ EC system (Antec) connected on-line to the high resolution mass …
Analysis of Protein-Phenolic Compound Modifications Using Electrochemistry Coupled to Mass Spectrometry
C Kallinich, S Schefer, S Rohn - Molecules, mdpi.com … Exemplarily, the electrochemical oxidation of chlorogenic acid was optimized by varying the voltage from 0.3 V to 3 V in steps of 0.3 V using a boron-doped diamond electrode and a Roxy ®potentiostat (Antec Scientific, Zoeterwoude, The Netherlands) …

27. Conclusions EC/MS in Drug Metabolism
Fast: minutes vs. weeks
No isolation/clean-up
Phase I and II metabolism
Complementary to in vivo and in vitro
Saving rodents (mice, rats)
Safety (no exposure to biohazard samples)
 EC/MS powerful tool for biomimetic oxidation 27