Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry.

INTRODUCTION: Anthocyanins are important plant secondary metabolites. They show strong antioxidant activities and have potential as anti-cancer agents. Viola yedoensis and V. prionantha are traditional Chinese medicines and ornamental plants. However, the anthocyanin compositions of these two species are still unresolved.

OBJECTIVE: To develop a rapid and reliable high-performance liquid chromatography (HPLC) method for the separation and identification of anthocyanins from V. yedoensis and V. prionantha.

METHODOLOGY: Samples were extracted in methanol-water-formic acid-TFA (70:27:2:1, v/v). HPLC analysis was done on a C(18) column (TSK-GEL ODS-80Ts: 150 × 4.6 mm i.d.). Four solvent systems were tested to optimise the separation of anthocyanins using different gradient separation systems. HPLC-photodiode array detection (DAD) coupled to electrospray ionisation mass spectrometry (ESI-MS) was used to carry out the comprehensive characterisation of anthocyanins.

RESULTS: Fourteen anthocyanins were characterised within 40 min with satisfactory peak resolution by a gradient composed of 10% aqueous formic acid and formic acid-acetonitrile-water (10:40:50, v/v). The calibration curve showed an excellent linear regression (r(2)  = 0.9995) and low intra- and inter-day variations (RSD < 3.67%). The detected anthocyanins derived from Dp, Cy, Pt, Mv and Pn, could be divided into three groups: non-acylated glycosides, acetylglycosides and coumaroylglycosides. Anthocyanins distribution exhibited remarkable differences in aglycone levels and acylation patterns.

CONCLUSION: The optimised method was successfully applied for the analysis of 14 anthocyanins from V. yedoensis and V. prionantha. The identification of anthocyanin constitutions is valuable for breeding and will open up new prospects for their medicinal application.