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

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.