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)(2)SO4 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)(2)SO4 concentration 22% for the ATPS, extraction temperature 80 degrees 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 (R-2 > 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 mu g/g and 94.14%-105.5%, respectively. The extraction process was explored by comparative study. The results demonstrated that addition of (NH4)(2)SO4 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. (C) 2016 Elsevier B.V. All rights reserved.