2 4 6 tris pyridyl s triazine

Authentic standards for L-ascorbic acid, lycopene, β-carotene, lutein, apigenin, kaempferol, myricetin, quercetin, rutin, (±)-naringenin, luteolin, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), and chemicals such as acetic acid, tert-butylhydroquinone (TBHQ), sodium carbonate, sodium nitrite, aluminum chloride hexahydrate, Folin-Ciocalteu reagent, sodium acetate trihydrate, 2,4,6-tris-(pyridyl)-s-triazine (TPTZ), HCl, formic acid, ferric chloride hexahydrate, and potassium dihydrogen phosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Likewise, metaphosphoric acid was obtained from Yakuri Pure Chemicals Co. (Uji, Kyoto, Japan). Acetonitrile (HPLC grade) and MeOH (HPLC grade) were purchased from Avantor Performance Materials Co. (Center Valley, PA, USA). Chloroform was obtained from Daejung Chemicals & Materials Co. (Siheung, Gyeonggi-do, South Korea).

The ferric reducing antioxidant power (FRAP) method measures the change in absorbance that occurs when the TPTZ (2,4,6-tris-pyridyl-S-triazine) (–Fe(III)) complex is reduced to the TPTZ-Fe2+ form in the presence of antioxidant compounds [47 (link)]. Briefly, the FRAP reagent was prepared by mixing 10 mM tris-pyridyl-S-triazine (TPTZ) solution in 40 mM HCl plus 20 mM FeCl3 and 0.25 M sodium acetate buffer (pH 3.6) in a volume ratio of 1 : 1 : 10. The acetate buffer used in the FRAP assay was prepared by dissolving 1.90 g/L of sodium acetate in water and adjusted to pH 3.6 with 16.0 mL/L glacial acetic acid. An aliquot (6 μL) of serum from adolescents stratified in three groups according to their polyphenol intake was mixed with 140 μL of FRAP reagent and 0.06 μL of H₂O. The absorbance of the reaction mixture was measured at 593 nm in Multiskan SkyHigh (Thermo Fisher Scientific, Waltham, MA, USA). A solution of FeSO₄ (1 mM) was used to obtain the calibration curve. 2,4,6-Tris-pyridyl-S-triazine (TPTZ), FeCl₃, FeSO₄, glacial acetic acid, and oleic acid were purchased from Sigma-Aldrich (Milan, Italy).