High-speed counter-current chromatography coupled online to high performance liquid chromatography–diode array detector–mass spectrometry for purification, analysis and identification of target compounds from natural products

• A novel online HSCCC–HPLC–DAD–MS system was developed.
• Two-step HSCCC was used to isolate target compounds.
• Key HSCCC effluent was analyzed online with shortened manipulation time.
• Five main flavonoids with purities over 99% were purified from M. doumeri.

A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC–HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC–HPLC–diode array detector–mass spectrometry (HSCCC–HPLC–DAD–MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC–DAD–MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4′,6′-dihydroxyhydrochalcone-2′-O-β-d-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25 mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products.