Microwave-assisted aqueous two-phase extraction coupled with high performance liquid chromatography for simultaneous extraction and determination of four flavonoids in Crotalaria sessiliflora L.

A novel method of simultaneous extraction and determination of vitexin, isovitexin, orientin and isoorientin in Crotalaria sessiliflora L. was developed by microwave-assisted aqueous two-phase extraction (MAATPE) coupled with high performance liquid chromatography (HPLC). With an aqueous two-phase system (ATPS) of ethanol/(NH4)2SO4 as extracting agent, the effects of the composition of the ATPS, extraction temperature, extraction time, particle size and solvent-to-material ratio on extraction yields of the flavonoids were investigated by single-factor experiment and response surface methodology (RSM). Optimum conditions were concluded as follows: ethanol concentration 32% and (NH4)2SO4 concentration 22% for the ATPS, extraction temperature 80 °C, extraction time 8 min, particle size 80 mesh and solvent-to-material ratio 50:1. With the help of MAATPE, HPLC analysis of four flavonoids above has been successfully conducted with UV detection at 340 nm with linear ranges (0.20-20.00 mg/L), correlation coefficients (R2 > 0.9996) and limits of detection (0.0309-0.0492 mg/L) using acetonitrile-water containing 0.5% phosphoric acid solution (14:86) as the mobile phase. Under the optimum conditions, MAATPE coupled with HPLC was applied to simultaneous determination of vitexin, isovitexin, orientin and isoorientin in Crotalaria sessiliflora L. Their extraction yields and recoveries were in range of 162.7-240.0 μg/g and 94.14%-105.5%, respectively. The extraction process was explored by comparative study. The results demonstrated that addition of (NH4)2SO4 in the water-ethanol mixture improved not only demixing effect but also microwave action, and extraction yields of the ATPS were superior to that of mono-phase solvent such as water, ethanol and the combination of both. Thus, MAATPE was a rapid and efficient alternative to the conventional extraction approaches for herb analysis and quality control.