Integration of magnetic solid phase fishing and off-line two-dimensional high-performance liquid chromatography–diode array detector–mass spectrometry for screening and identification of human serum albumin binders from Radix Astragali

•HSA binders were screened by Fe3O4@SiO2–HSA nanoparticles.•2D HPLC–DAD–MSn was developed to analyse and identify HSA binders.•Forty-seven active compounds were screened and identified.•Isoflavonoids and astragalosides were the main active classes in Radix Astragali. Radix Astragali is one of the most popular traditional medicinal herb and healthy dietary supplement. Isoflavonoids and astragalosides are the main bioactive ingredients. However, the systematic bioactive component analysis is inadequate so far. Then a facile method based on Fe3O4@SiO2–human serum albumin (Fe3O4@SiO2–HSA) magnetic solid phase fishing integrated with two-dimensional high-performance liquid chromatography–diode array detector–mass spectrometry (2D HPLC–DAD–MSn) was developed to fish out and identify HSA binders from Radix Astragali. The immobilized HSA displayed a high stability with 96.2% retained after ten consecutive cycles. 2D HPLC system (size exclusion chromatography×reversed phase chromatography, SEC×RP) were developed and optimised. Forty-seven bioactive compounds including thirty-four isoflavonoids and thirteen astragalosides were screened and identified or tentatively deduced based on their retention time, ultraviolet (UV), accurate molecular weight and diagnostic fragment ions. The results indicated that the integrated method could be widely applied for systematical fishing and identification of bioactive compounds, especially for low-abundance and overlapped compounds, from complex mixtures.