*4.2. Qualitative and Quantitative Analysis of Floral Pigments*

Previously frozen petal tissues were ground into fine powder in liquid nitrogen. Next, 200.0-mg portions of individual petal tissue samples were placed in centrifuge tubes containing extraction reagent (70:27:2:1 (*v*/*v*/*v*/*v*) methanol-water-formic acid-trifluoroacetic acid) [85] and vortexed for 1 min at room temperature followed by ultrasonic extraction in a KQ 2200B ultrasonic cleaner (Jiangsu, China) at 4 ◦C for 15 min. After storage overnight at −20 ◦C, the mixtures were centrifuged (A-14C, Sartorius, Goettingen, Germany) for 10 min at 12,000 rpm and 4 ◦C. Supernatants were collected and filtered through 0.22-µm nylon membranes [86] for high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis.

Preliminary HPLC analysis [86] was performed on a Waters 2695 system (USA) equipped with a W2996 photodiode array and a C18 column (5 µm, 4.6 × 250 mm i.d.; WondaCract ODS-2, Shimadzu, Shanghai, China). The major parameters were set as follows: column temperature = 30 ◦C, absorption spectrum = 200 to 600 nm, injection volume = 10 µL, and flow rate = 0.8 mL/min. Gradient separation was carried out using a two-solvent system, 0.5% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B), as follows: 0 min, 5% B; 5 min, 10% B; 20 min, 20% B; 30 min, 25% B; 33 min, 10% B; 35 min, 5% B; and 50 min, 5% B. Quantification of WT and RT samples was performed using three replicates (WT1, WT2 and WT3, and RT1, RT2 and RT3, respectively) and an external standard. The external standard was cyanidin 3-*O*-glucoside chloride (Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in the same extraction reagent (70:27:2:1 (*v*/*v*/*v*/*v*) methanol-water-formic acid-trifluoroacetic acid) with a concentration of 0.125 mg/mL.

Following preliminary HPLC identification, anthocyanins were detected using an HPLC system (Agilent 1200LC, Santa Clara, CA, USA) equipped with a diode array detector at 520 nm and the same C18 column used above and coupled to an electrospray ionization-mass spectrometer (ESI-MS) (6310 MSD Trap VL, Agilent, USA). ESI-MS was performed with the following settings: positive ionization mode (ESI, *m*/*z* 50–1000 mass units), gas temperature = 350 ◦C, flow rate = 8.0 L/min, nebulizer pressure = 35 psi, and capillary exit voltage = 120.4 V.
