A new HPTLC-image analysis approach for assessing interactions and antioxidant profiles of adrenergic drugs

This study proposes a new approach for the in-vitro evaluation of antioxidant profile of adrenergic drugs using high-performance thin-layer chromatography coupled with image analysis techniques (HPTLC-IA). In this regard, stationary phases of different polarity (Silica gel and RP-8, RP-18 W, CN and DIOL) were selected to simulate different environments and interactions that adrenergic drugs might encounter in the human body. Antioxidant activity was quantified using both DPPH^• and ABTS^⁺• radical scavenging assays on all five stationary phases. Image processing and analysis was used to quantitatively compare the free radical scavenging potential of the analyzed compounds after converting the RGB image into different color channels, namely the green channel for the DPPH test, and the red channel for ABTS. Chromatographic data were processed using multivariate analysis. Results revealed that highly polar compounds, that is norepinephrine, etilefrine, metaraminol and midodrine exhibited strong interactions with silica gel but limited retention on reversed-phase, while lipophilic derivatives, namely naphazoline, xylometazoline, clenbuterol and bambuterol demonstrated strong affinity for non-polar and moderately polar phases, predicting good membrane penetration and blood-brain barrier permeability. Interestingly, some β-agonists (bambuterol, fenoterol, buphenine and irsoxsuprine) with polar groups showed unexpectedly weak silica gel retention, highlighting their dominant lipophilic contributions. Regarding the antioxidant activity, the results showed that phase interactions with the stationary phase significantly influenced the activity of adrenergic drugs. Thus, the polar phases (silica gel, DIOL) enhanced radical scavenging activity, whereas non-polar phases often reduced it. The developed HPTLC-IA method, integrating selective stationary phases and two-radical assays, offers a novel and cost-effective approach for screening the possible variation of antioxidant activity of drugs after their interaction with physiological media constituents.