1. Mimicking Oxidative Metabolism “OxMet” using ROXY EC System
ASMS 2015
St. Louis, MO, USA

2. Outline I. Introduction - Drug metabolism – what you should know
- Case Study 1 – Acetaminophen
- Case Study 2 – Amodiaquine
- Examples from Industry
II. 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
Adapted from 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 Chroamtography 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. Conclusions EC/MS in Drug Metabolism
ionization techniques: ESI (+/-) API, APCI
weeks vs. seconds
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 25