Microemulsion liquid chromatographic method for simultaneous separation and determination of six flavonoids of Apocynum venetum leaf extract.

A simple, cost-effective, and efficient method was developed for the rapid simultaneous separation and determination of six flavonoids (rutin, hyperoside, quercetin-3-O-sophoroside, isoquercitrin, astragalin and quercetin) of Apocynum venetum leaf extract by reversed phase high performance liquid chromatography using a microemulsion system mixture as the mobile phase. Separations were performed on the Zorbax Extend-C18 column with UV detection at 360nm. The flow rate was 0.8mLmin(-1). The optimized microemulsion mobile phase consisted of 2.5% (v/v) n-butanol, 1.2% (v/v) of Genapol X-080, 0.5% (v/v) ethyl acetate and 95.8% (w/v) of aqueous 20mM phosphoric acid, pH adjusted to 6.0 with 0.3% triethylamine. Under the optimized conditions, the calibration curve for six flavonoids was linear in the range of 5-1000μgmL(-1) with the correlation coefficients greater than 0.9994. The intra-day and inter-day precision (RSD) were below 8.11% and the limits of detection (LOD) for the six flavonoids were 1.7-6.0μgmL(-1) (S/N=3). The microemulsion liquid chromatography (MELC) method was successfully applied to separate and determine the six flavonoids of A. venetum leaf extract.