Identification of Anthocyanins by HPLC-MS/MS

Anthocyanin accumulation could accumulated in both leaves and ray florets of cineraria. In this study, the leaves and ray florets of VeB was used to identify anthocyanin components by HPLC-MS/MS analysis, and the ray florets of VeW and VeC were used as the control. Anthocyanin extraction and determination were performed as previously described (Sun et al., 2010). A total of 0.1 g of the sample was finely ground into powder in liquid nitrogen, and then homogenized in 5 ml of anthocyanin extracts [methanol: water: formic acid: trifluoroacetic acid (70:27:2:1, v/v/v/v)], leached at 4°C for 24 h in the dark. The supernatant volume was passed through a 0.22 μm filter after centrifugation. The sample was determined by the DIONEX high performance liquid chromatography equipped with a P680 HPLC pump, UltiMate 3000 auto-sampler, Thermostatted Column Compartment-100, and a TSK-GEL ODS-80Ts QA column (4.6 mm × 150 mm). The loading volume was 20 μL, mobile phase A was methanol:acetonitrile = 15:85 (v/v), mobile phase B was formic acid: water = 10:90 (v/v), column temperature was 25°C, flow rate was 1 mL/min. The gradient elution was performed as follows (A%/B%): 0–20 min, 70%–47%/30%–53%; 20–40 min, 47%/53%; 40–45 min, 47%–70%/53%–30%; 45–60 min, 70%/30%. Meanwhile, HPLC-ESI-MS/MS was used to analyze the anthocyanin structure of typical samples by Agilent 1100 LC/MSD Trap VL liquid chromatography-tandem mass spectrometry. The conditions of liquid chromatographic analysis were the same as above. The retention time and peak area of each sample was measured at wavelengths of 520 nm and in comparison with standards as cyanidin chloride (Sigma). Mass spectrometry analysis of each pigment components was mainly compared with the data of previous study (Sun et al., 2010).