2.4.2. High-Performance Liquid Chromatography with a Diode Array Detector (HPLC-DAD)

For HPLC-DAD analysis, *Reseda luteola* plant was extracted by placing 1 g of the dry plant material (as collected from nature) with 100 mL of methanol:water (70:30, *v*:*v*) and heating in a water bath at 60 ◦C for one hour, as described in [26]. The dye from lake pigments was extracted by placing in an eppendorf, 10 mg of powder with a 1 mL solution of oxalic acid (0.2 M):methanol:acetone:water (0.1:3:3:4, *v*:*v*), as described in [27].

Prior to HPLC-DAD analysis, all extracts were centrifuged at 12,000 rpm for about 10 min. The supernatant liquid was gently removed and filtered through a 0.45 μm filter. Before analysis, the solution was diluted with methanol:water (70:30, *v*:*v*) if necessary.

The analysis was carried out in a Thermofinnigan Surveyor® HPLC-DAD system with a Thermofinnigan Surveyor PDA (Thermofinnigan, San Jose, CA, USA), an autosampler, and a gradient pump. The sample separations were performed in a reversedphase column, RP-18 Nucleosil column (Macherey-Nagel) with 5 μm particle size column (250 mm × 4.6 mm), with a flow rate of 1.7 mL/min with the column at a constant temperature of 35 ◦C. The samples were injected via a Rheodyne injector with a 25 μL loop. The elution gradient consisted of two solvents, A: methanol and B: 0.1% (*v*/*v*) perchloric acid aqueous solution. A gradient elution program was used of 0–2 min isocratic 7% A, 2–8 min linear gradient to 15% A, 8–25 min linear gradient to 75% A, 25–27 min linear gradient to 80% A, 27–29 min linear gradient to 100% A, and 29–30 min isocratic 100% A (10 min re-equilibration time). The eluted peaks were monitored at 350 nm.

The peaks were integrated and the area of each peak was recorded as well as the percentage. Peak area calculation was done by defining the time intervals for each peak. The area below the peak was integrated within this interval is measured and the percentage of each area is calculated by dividing by the sum of all the peak areas. For this analysis, it was considered the area of the nine peaks visible at λ = 350 nm, between 14 and 24 min, as shown in Figure 2.

**Figure 2.** Chromatogram of an extract in MeOH:H2O (70:30, *v:v*) of *Reseda luteola*, λ=350 nm: (1) apigenin-6,8-di-*C*-glucoside, (2) luteolin di-*O*-glucoside; (3) luteolin 3 ,7-*O*-glucoside; (4) luteolin 7-*O*-glucoside, (5) apigenin 7-*O*-glucoside, (6) chrysoeriol glycoside, (7) luteolin 4 -*O*-glucoside, (8) luteolin, (9) apigenin.
