2‑Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3‑Oxide (PTIO^•) Radical Scavenging: A New and Simple Antioxidant Assay In Vitro

Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent results. The study develops a new antioxidant assay using the 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO^•). After the investigation of various factors, the experimental protocol was briefly recommended as follows: PTIO^• and the sample solution were added to phosphate buffer (pH 7.4, 50 mM), incubated at 37 °C for 2 h, and then spectrophotometrically measured at 557 nm. The validation test based on 20 pure compounds and 30 lyophilized aqueous extracts suggested that PTIO^• scavenging had a good linear relationship, stability, and reproducibility. In the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, PTIO^• was observed to give m/z 234 when encountering l-ascorbic acid. As an antioxidant assay, PTIO^• scavenging possesses four advantages, i.e., oxygen-centered radical, physiological aqueous solution, simple and direct measurement, and less interference from the tested sample. It can also satisfactorily analyze the antioxidant structure–activity relationship. PTIO^• scavenging has no stereospecificity and is at least involved in H⁺ transfer.