1. Pharmaceutical Stability Testing Using Electrochemistry/MS ASMS 2015 St. Louis, MO, USA

2. Outline Drug Stability & Degradation - Naltrexone a case study Antioxidants Redox capacity Electrochemical Synthesis Conclusions

3. Purposeful Degradation / Drug Stability Where does Electrochemistry/MS fit in? EC/MS offers massive advantages in comparison to peroxide/acid purposeful degradation and stability testing trials: - Controllable - Rapid - Generates reference materials

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

5. Naltrexone 0.8 1.0 5 x10 1000 2000 Intens. 200 400 600 Intens. 2000 Intens. 0 1 4 x10 Intens. 2000 4000 Intens. 0 500 1000 Intens. 12345678Time [min] 00.751.52.253.0volts 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) C20H23NO6 Dehydrogenation Dimerisation Dehydrogenation+hydroxylation hydroxylation Dihydroxylation Trihydroxylation MS Infusion: voltage ramping 0 – 3.0 V in 7 min Substrate

6. EC-UHPLC-UV-ESI-TOF-MS of Naltrexone API Solutions A B Trihydroxy-NLT isomers 2,2-Bis(naltrexone) C40H44N2O8 MW = 680 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. 10-hydroxynaltrexone C20H23NO5 MW = 357

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

8. 8 Study of Anti-Oxidant Performance EC-MS: Infusion mode (DC) The 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. The addition of BHT to the solution 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.

9. 9 Anti-Oxidative Performance of Butylated hydroxytoluene (BHT) on Oxycodone (OXY) C 18 H 21 NO 4 Mr = 315

10. Electrochemical Synthesis 10 - Astra Zeneca - Novartis - Sandoz - Pfizer - Merck - Roche - BASF - DSM - Novo Nordisk - Sanofi Aventis - Bayer - J&J - etc. mg quantities metabolites degradation products REDOX products 80 mL bulk cell

11. Electrochemical Synthesis 1. Potential based fractionation in vial(s) e.g., 2400 mV for 394 and 520 metabolite 2. Inject in HPLC (semiprep) 3. Fractionation/purification 4. ID by NMR 2400 mV 11

12. t = 0 t = 15 min t = 30 min after synthesis experiment SynthesisCell™

13. Conclusions Pharmaceutical Stability Testing EC/MS is a powerful platform for - fast drug stability and purposeful degradation studies - easier identification of degradation products - direct measurement of antioxidant’s capacity Electrochemical synthesis - fast and cost effective synthesis of mg quantities of degradation products - complementing wet chemical synthesis - sufficient reference material for NMR and MS