Totalchrom workstation software

A Perkin Elmer Series 200 HPLC system, equipped with an autosampler, binary pump and UV/Vis detector (all of Series 200), was applied with a TotalChrom Workstation software (PerkinElmer, Waltham, MA, USA).
Chromatographic separations were performed on a Restek Ultra IBD C18 column (5 µm particle size, 250 × 4.6 mm i.d.) with Ultra IBD guard column (5 µm particle size, 10 × 4 mm i.d.), Restek, Bellefonte, PA, USA.

High-performance liquid chromatography (HPLC) measurement of flavonols were carried out using the Perkin Elmer HPLC system (Waltham, Massachusetts, USA) consisting of a binary pump Series 200, an autosampler, Peltier column oven Series 200, UV-VIS detector Series 200, and UltraAqueous C18 column (250 × 4.6 mm, Resek, Bellefonte, PA, USA). TotalChrom Workstation software (version 6.2.1, Perkin Elmer), was used to process the chromatographic data. After filtration through a 0.45 µm syringe filter, the extract was injected directly through a 20 µL fixed loop into a guard of the C18 column. Each sample was injected three times in order to check its reproducibility. A gradient consisting of solvent A (0.2% H₃PO₄) and solvent B (MeOH/acetonitrile, 1:1 v/v) was applied at a flow rate of 0.8 mL/min as follows: 0–0.5 min 96% A and 4% B; 0.5–40 min 50% A and 50% B; 40–45 min 40% A and 60% B; 45–60 min 0% A and 100% B; 60–68 min 0% A and 100% B; 68–70 min 96% A and 4% B; 70–80 min 96% A and 4% B. Detection of the elution peaks was at λ = 360 nm. Flavonoid compounds were identified on the basis of their retention times and quantified using external standard calibration curves. Standards for identification purposes were: quercetin, quercetin 4′-monoglucoside, and quercetin 3,4′-diglucoside prepared in methanol. The resultant concentrations are expressed as mg/100 g of dry weight.