Analysis and improved characterization of minor antioxidants from leaves of Malus doumeri using a combination of major constituents’ knockout with high-performance liquid chromatography–diode array detector–quadrupole time-of-flight tandem mass spectromet

• Major constituents’ knockout + HPLC–DAD–QTOF-MS/MS was developed.
• Five major compounds in M. doumeri were knocked out by HSCCC to enrich minor ones.
• DPPH–HPLC was established to screen minor antioxidants.
• HPLC–DAD–QTOF-MS/MS was developed to identify thirty minor antioxidants.
• Twenty-six antioxidants were reported in M. doumeri for the first time.

Due to the complexity of natural products, efficient identification of bioactive compounds, especially for minor compounds, would require a huge effort. Here, we developed an effective strategy based on combining major constituents’ knockout with high-performance liquid chromatography–diode array detector–quadrupole time-of-flight tandem mass spectrometry (HPLC–DAD–QTOF-MS/MS) to comprehensively identify minor antioxidants in Malus doumeri, one of the longest known and most used tonic plant in Taiwan. First, five major compounds (I–V) in M. doumeri were knocked out by two-step stepwise high-speed countercurrent chromatography (HSCCC). Second, minor antioxidants were screened by 1,1-diphenyl-2-picrylhydrazyl radical–HPLC (DPPH–HPLC) assay. Third, structures of thirty minor antioxidants, including 11 dihydrochalcones, 4 flavanones, 3 flavonols, 2 flavones, 3 aurones and 7 phenolic acids, were unambiguously or tentatively identified by matching their characteristic UV spectra, accurate mass signals and key diagnostic fragment ions with standards or previously reported compounds. Twenty-six of them, as far as was known, were discovered from M. doumeri for the first time. The results indicated that the proposed method was a useful approach to explore minor bioactive compounds from complex natural products.