1. Pharmaceutical Stability Testing using Electrochemistry-MS
ASMS 2018
San Diego, CA, USA

2. Outline Introduction to Electrochemical (EC) degradation
2 Case studies: Naltrexone & MDC
Conclusion EC as stress test
Antioxidant efficacy
Excipient stability
Overall conclusions

3. Principle of Electrochemical Oxidation
Potential (∆E) is the driving force of the reaction
∆E (Potential)
Reduction Oxidation
We have here a reaction cell
It consists of 3 electrodes WE
Ref
Aux to avoid polarisation effects, and allows to work under better defined and controlled conditions
Potential difference between the WE and Ref,
By this we induce oxidation or reduction depending on the potential applied here.
The reaction is monitored by Mass spectrometry
To that end, we use a potentiostat called ROXY shown here on the right
It is fitted with a reaction cell, an exemple of is shown here called a micro Prep Cell
For every flow rate, we have an appropriate flow-through cell
We have 3 different flow-through cells,
No details about the synthesis cell
Besides varying the voltage we can further vary selectivity by using different WE:
BDD GC Ti Au

4. Instrumental Setup EC-MS
Infusion
EC-MS
Pre-separation
EC-LC-MS
Where can we place this reactor cell.
There are 3 basic setups combining electrochemistry to liquid chromatography and mass spectrometry.
The simplest setup in which to use the ROXY consists of a potentiostat for reactions control and a syringe pump delivering the analyte through the cell – where reactions take place – to the MS where the reaction products are detected.
In case of complex mixture, The ROXY can also be positioned in front of the LC-MS, to generate products that will be subsequently separated and detected.
Deconvolute sample complexity
It can also be positioned between the LC and the MS, to perform a reaction prior to detection by MS
for example for the online reduction of S-S bridges in proteins.
In our case today, I will show data obtained with the top two setups

5. Purposeful Degradation / Drug Stability
Case Study 1 – Naltrexone (Pfizer)
opiate (morphine)
blocks addiction
short shelf-live oxidation (hydroxylation)

6. Naltrexone MS Infusion: voltage ramping 0 – 3.0 V in 7 min 2.0 V 0 V
0.8
1.0 5
x10
1000
2000
Intens.
200
400
600 1 4
4000
500 2 3 6 7 8
Time [min]
0.75
1.5
2.25
3.0
volts
NLT -
dimer (m/z = 341)
C40 H44N208
NLT[
2H] (m/z = 340)
C20H21NO4
Naltrexone API (m/z = 342)
C20H23NO4
NLT+14 (m/z = 356)
C20H21N05
NLT+16 (m/z = 358)
C20H23N05
NLT+32 (m/z = 374)
C20H23NO6
NLT+48 (m/z = 390)
Dehydrogenation
Dimerisation
Dehydrogenation+hydroxylation
hydroxylation
Dihydroxylation
Trihydroxylation
MS Infusion: voltage ramping 0 – 3.0 V in 7 min
Substrate
2.0 V
0 V
active pharmaceutical ingredients (API) solutions

7. EC-HPLC-UV-MS of Naltrexone API Solutions
UV chromatograms of A: aged naltrexone HCl standard and B: naltrexone HCl standard oxidised by EC at 1.3V in DC mode showing base-peak mass assign-ments from TOF-MS
Hydroxylated, dehydrated and dimerised degradant product peaks were identified and concentrations of the “real world” degradants observed API in the aged standard were increased significantly facilitating structure elucidation by
HR-MS-MS. B
API Solutions (Active Pharmaceutical Ingredients)
2,2-Bis(naltrexone)
C40H44N2O8 MW = 680
Trihydroxy-NLT isomers
10-hydroxynaltrexone
C20H23NO5 MW = 357

8. Drug Stability / Purposeful Degradation
What else did we do in the 2 days at Pfizer?
All known oxidation products found and re-confirmed
4 Additional new compounds found (proprietary)
Collected enough material for LC-MS studies offline

9. Case Study 2: MDC
Chemical structure of model drug compound ((2S,3S)-2-(diphenylmethyl)-N-[2-methoxy-5-(propan-2-yl) benzyl]-1azabicyclo[2.2.2]octan -3-amine)

10. Oxidation Products of MDC
Compounds 2–6: accelerated stability studies (). Compounds 3 and 4 both by: forced degradation (#) & electrochemically (). Compound 6 and 16 only electrochemically. Chemical structures for compounds 3, 4, 4’, 6, 7, 8 and 16 were derived on the basis of exact masses, and MS/MS fragmentation patterns.

11. Forced Degradation
LC–UV chromatogram at 225 nm. Samples after 3 days of challenge condition: (A) 0.3% H2O2, (B) 5 mM AIBN; 2,2’-Azobis (2-methy-lpropionitrile), radical initiator

12. LC–UV chromatograms at 225 nm for electrochemically oxidized samples at 1200 mV at pH = 3.9, 7.1, Peak 1 drug substance.
S. Torres et al., J. of Pharm. and Biomedical Analysis 115 (2015) 487

13. Generated Oxidation/Degradation Products
Oxidation products generated via pharmaceutically relevant stress testing (ST), electrochemically (EC) and predicted by in silico (computational).

14. Electrochemical mechanism for the secondary amine cleavage and corresponding formation of oxidation products 3, 4. 4, 7 and 8.
S. Torres et al., J. of Pharm. and Biomedical Analysis 115 (2015) 487

15. Fully Automated on-line EC-LC-UV-MS System
S. Torres et al., J. of Pharm. and Biomedical Analysis 131 (2016) 71

16. On-line EC-LC-UV-MS with Cleaning Step
No cleaning
Automated
cleaning
S. Torres et al., J. of Pharm. and Biomedical Analysis 131 (2016) 71

17. ThP829, 10:30 am - 2:30 pm

18. Conclusion EC as Stress Test
Electrochemistry as an oxidative stress testing method offers
No need of storage/disposal of strong oxidizing agents  green
To study reactions in  real-time
The ability to select optimum experimental conditions for generation of the desired oxidation products profile(s)  tunable
High degree of  automation
Generates a high number of structurally-related oxidation products not recorded in traditional stability studies  new drug candidates?

19. Outline Introduction to Electrochemical (EC) degradation
2 Case studies: Naltrexone & MDC
Conclusion EC as stress test
Antioxidant efficacy
Excipient stability
Overall conclusions

20. EC-MS: Infusion mode (DC)
Real Time Study of Antioxidant Efficacy
EC-MS: Infusion mode (DC)
Effectiveness of butylated hydroxytoluene (BHT) as an anti-oxidant to stabilise oxycodone (OC) in solution was studied by EC-MS and EC-LC-UV-MS. BHT significantly increased the OC molecular ion base peak intensity and reduced the yield of oxycodone degradant peaks produced when voltage was applied to the EC cell. The fine voltage / reaction control offered by the EC-MS system allowed reactions to be studied rapidly in real time.

21. Effectiveness of BHT-Antioxidant on Oxycodone Stability
C18H21NO4
Mr = 315

22. Excipient Stability
Glucose oxidises in solution to form the potentially genotoxic impurity (PGI) 5-HMF, which then further oxidises when autoclaved. Oxidation of glucose could be controllably studied by EC-MS and the degradant peak profiles from an autoclaved glucose solution closely matched those observed by EC-HR-MS
Davis, S. E., B. N. Zope, et al. (2012). Green Chemistry 14(1) (2012) 143

23. Study of Excipient Oxidation by EC

24. Overall Conclusions Electrochemistry/MS is a powerful platform for:
Rapid drug stability testing and purposeful degradation
Easier identification of degradation products
Furthermore it allows for:
Direct measurement of antioxidant efficacy
Stability and interference testing of excipients
Rapid optimization of galenic formulation 24