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

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

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

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

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

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

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

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

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)

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.

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  

LC–UV chromatograms at 225 nm for electrochemically oxidized samples at 1200 mV at pH = 3.9, 7.1, 8.8. Peak 1 drug substance. S. Torres et al., J. of Pharm. and Biomedical Analysis 115 (2015) 487

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

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

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

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

ThP829, 10:30 am - 2:30 pm

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?

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

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.

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

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

Study of Excipient Oxidation by EC

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