Application of Liquid Chromatography in the Study of Stability and Degradation of Pharmaceutically Important Materials

Nowadays, the determination of free radicals has become one of the main goals in chemical industry, especially in pharmaceutics, because of the reactivity of radicals and thus the formation of impurities and possibly inactive or toxic products resulting from reactions with active pharmaceutical ingredients. This work focuses on the determination of radicals contained in pharmaceutical excipients using high-performance liquid chromatography on reverse phase (RP-HPLC). As a desired result, we are looking for a radical scavenger that is able to undergo a defined reaction with the radical and linearly produce a quantifiable product. The stable radical molecule (4‑hydroxy-2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) is used as a standard of radical behavior. Firstly, the purity of the radical was verified for possible impurities by MS analysis, which showed a high purity of TEMPO. Secondly, the efficiency of several well-known scavengers was tested by reaction with TEMPO. While the reduced glutathione showed a very fast reaction with the radical, the predicted product (in this case oxidized glutathione) did not form linearly, and the subsequently performed MS analysis showed multiple reaction pathways, mostly explained by breakage of the peptide chain. Coumarin was used next in order, which showed no determinable reaction with the radical at all, even after increasing the temperature. At present, the main attention is focused on ascorbic acid and its oxidation product dehydroascorbic acid. We expect the scavenging mechanism to be very similar to that of reduced glutathione, however, the possibilities of side products are limited, the main issue at this point being the RP-HPLC separation of ascorbic and dehydroascorbic acid, respectively.