Anthocyanin Profiling by HPLC-MS

An Agilent 1100 HPLC system coupled with an MSD Trap VL ion-trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used for the analysis of anthocyanins [19 (link),38 (link)]. A Kromasil-C18 column (250 × 4.6 mm, 5 µm) was used for the separation of anthocyanins. The mobile phase consisted of (A) 6% (v/v) acetonitrile containing 2% (v/v) formic acid, and (B) 54% (v/v) acetonitrile containing 2% (v/v) formic acid. The column was set at 50 °C and the flow rate was 1.0 mL/min. A sample volume of 30 µL was injected to the system. The gradient was as follows: 0–1 min, 10%B; 1–18 min, 10%B to 25%B; 18–20 min, 25%B isocratic; 20–30 min, 25%B to 40%B; 30–35 min, 40%B to 75%B; and 35–40 min, 70%B to 100%B. The wavelength for the detection was set at 525 nm. Positive electrospray ionization was used with the nebulizer pressure of 35 psi, the temperature of +325 °C, and the dry gas flow rate of 10 mL/min. A full scan mode from m/z 100 to 1500 was recorded. Anthocyanins were tentatively identified by comparing their mass spectrum with the references [39 (link),40 (link)]. The external standard malvidin-3-O-glucoside was used for the quantitation of anthocyanins.