Quantification of Flavonoids and Phenolic Acids in Hemerocallis Extracts

Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for qualitative and quantitative analysis of flavonoids and phenolic acids in Hemerocallis extracts. The separation of analyzed compounds, injected in a 3-µL amount, was performed on a Zorbax SB-C₁₈ analytical column (2.1 × 100 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA) at 25 °C. Elution was carried out using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0–2 min—20% B, 3–4 min—25% B, 5–6 min—35% B, 5–6 min—35% B, 8–12 min—65% B, 14–16 min—80% B, 20–28 min—20% B. The flow rate was 300 µL/min. The mass spectra of analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 °C, nebulizer gas 50 psi, curtain gas 30 psi, source voltage −4500 V for phenolic acids and flavonoid glycosides, and capillary temperature 550 °C, nebulizer gas 30 psi, curtain gas 20 psi, and source voltage −4500 V for flavonoid aglycones analysis. Details of LC-ESI-MS/MS analysis are presented in Table 1 and were described in our previous research [41 (link)]. The Analyst 1.5 software (AB Sciex, Redwood City, CA, USA) was used for analysis and data acquisition.