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A rapid method for simultaneous extraction and separation of multiple alkaloids from Radix Sophorae Tonkinensis (RST) was developed by microwave-assisted aqueous two-phase extraction (MAATPE) using the aqueous two-phase extraction system (ATPS) of ethanol/Na 2 HPO 4 as extraction solvent. The effects of key factors on MAATPE extraction of alkaloids from RST were investigated by single-factor experiment and response surface methodology (RSM). The optimum conditions were concluded as follows: the phase ratio of 2.60 for the ATPS, the particle size of 100 mesh, the liquid-to-material ratio of 75:1, extraction temperature of 90 C and extraction time of 5 min. The RSM model was significant (p<0.0001) and adequate for prediction of process efficacy, and the experimental yield of 29.410.21 mg/g for alkaloids under optimum conditions was very close to the predicted value. Compared to conventional extraction methods, alkaloids were extracted preferentially from RST to top phase by MAATPE with higher yield and shorter extraction time. By means of high-resolution ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap/MS) and HPLC with UV detection, identification and quantification of nine alkaloids extracted were accomplished. Matrine, sophocarpine, oxymatrin, sophoranol, oxysophocarpine, 5α-hydroxysophocarpine, sophoridine, cytisine and N-methylcytisine in RST were successively determined in range of 0.49310.284 mg/g with RSD's of 0.82.1%. Moreover, MAATPE mechanism was explored by means of the different extraction systems and scanning electron microscopy.Significant differences in extraction yield and cell rupture exhibited that the addition of the salt in the mixed solvents not only improved thermal effect and demixing effect, but also accelerated the mass transfer process and biphasic extraction capacity. MAATPE integrated the advantages of microwave-assisted extraction (MAE) and aqueous two-phase extraction (ATPE). Hence, it was also proved as a green, efficient and promising alternative to extraction of alkaloids from RST.

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