Extraction and UPLC-MS/MS analysis of flavonoids from putative flavonoid mutants

To determine the flavonoid composition of putative color mutants, extracts were prepared from lyophilized and ground leaf tissue, using the method described in¹⁸. In short, leaves were kept at −80 °C prior to lyophilization. Freeze-dried leaves were ground to a fine powder with a mortar and pestle. 50 or 100 mg lyophilized leaf powder was placed in a 15 ml plastic tube, protected from light, then, respectively, 1.5 ml or 3 ml solvent (methanol/water/acetic acid; 85:14.5:0.5 v/v), was added. The leaf powder was vortexed with the solvent for 30 sec, sonicated for 5 min, vortexed for another 30 sec, kept for 10 min at room temperature, and centrifuged at 1700 rcf for 5 min. The supernatant was decanted, then the extraction was repeated twice, and the decanted extracts pooled. The decanted solution was centrifuged at 1700 rcf for 8 min and filtered through 0.45 µm polytetrafluoroethylene (PTFE) filters (Fisher Scientific) for UPLC-MS/MS analysis.

Extracts were separated and analyzed by a UPLC-MS/MS system using the protocol described in⁷⁸. Since this protocol results in the co-elution of chlorogenic acid and cyanidin 3-O-malonylglucoside, we used a modified gradient elution to separate these two compounds. For this protocol, the mobile phase consisted of two components: Solvent A (0.5% ACS grade acetic acid in double distilled de-ionized water, pH 3–3.5), and Solvent B (100% acetonitrile). The initial conditions of the gradient were 95% A and 5% B; for 20 minutes the proportion reached linearly 80% A and 20% B. Within the next 3 minutes the proportion was 5% A and 95% B, which was maintained for 4 minutes. Within the following 3 minutes the gradient was adjusted to initial conditions, and 5 additional minutes were included for equilibration before subsequent injections.