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Direct evidence of 2-cyano-2-propoxy radical activity during AIBN-based oxidative stress testing in acetonitrile–water solvent systems



Oxidative susceptibility testing was performed on a drug substance containing a methoxy-naphthalene moiety. 2,2′-azobisisobutyronitrile (AIBN) was employed to initiate peroxy radical oxidation to mimic autoxidation processes. In acetonitrile (ACN)–water solvents, three major degradation products are formed. However, addition of small amounts of methanol to the solvent system completely eliminated the observed degradation products. To understand this effect, the structures of the three degradants have been elucidated using nuclear magnetic resonance, liquid chromatography–tandem mass spectrometry, and accurate mass Fourier transform ion cyclotron resonance mass spectrometry. One degradant structure definitively proves the degradation resulted from alkoxy radicals (2-cyano-2 propoxy radical) arising from the disproportionation of the tertiary AIBN-derived peroxy radicals, rather than from the intended action of the AIBN peroxy radicals themselves. The reaction occurs over a wide range of AIBN and drug substance concentrations. This “protective effect” of several percent methanol by volume is rationalized by known methanol H atom donation rates to similar tert-butoxy and cumyloxy radicals (ca. 10 M−1 s−1) and the high methanol concentration relative to the dilute substrate being investigated. This work confirms recent proposals for addition of at least about 10% methanol to the standard ACN–water AIBN stress testing diluent to insure that only the desired peroxy radical activity is present during the oxidative stress test. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1554–1568, 2013