Cyanidin 3 rutinoside

The individual ACNs in colored rice bran were quantified with HPLC and identified using ESI-MS/MS. Rice bran was extracted with acidified methanol as described above. The supernatant obtained after centrifugation was concentrated under nitrogen. The concentrate was centrifuged (10,000 g, 20 minutes, 4°C) and added with acidified methanol to make a total volume of 5 mL. This extract solution was diluted appropriately with 1% HCl aqueous solution, passed through a 0.45-μm Nylon filter, and 20 μL of the sample was injected to HPLC system for analysis. The HPLC system was equipped with a Hitachi L-6000 pump (Hitachi Ltd), a YMC-pack ODS-AQ column (250 mm × 4.6 mm, 5-μm particle size; YMC Co. Ltd., Kyoto, Japan), and a PDA detector (Hitachi Ltd.). The eluent was composed of 1% formic acid (A) and methanol (B). The ACNs were carried out at a flow rate of 1.0 mL/min by a gradient elution as follows: 0 minutes 25% B; 5 minutes 25% B; 9 minutes 33% B; 17 minutes 90% B; 18 minutes 100% B; 23 minutes 100% B. The chromatogram at the wavelength of 510 nm was used for quantification of ACNs. The ACNs separated using HPLC were collected and injected to the ESI-MS/MS system (Thermo Finigan LXQ, San Jose, CA, USA) for confirmation. The standards of Cy 3-glc, cyanidin 3-rutinoside, and peonidin 3-glucoside (Pn 3-glc) purchased from Extrasynthese Co. (Genay Cedex, France) were used for analyses.