Separation and detection of minor constituents in herbal medicines using a combination of heart-cutting and comprehensive two-dimensional liquid chromatography

Herbal medicines contain a large number of minor constituents, which could contribute to their therapeutic effects and provide valuable lead compounds for drug discovery. However, to explore minor constituents from complicated herbal extracts is usually laborious and time-consuming. In order to discover minor novel herbal constituents efficiently, we combined heart-cutting and comprehensive two-dimensional liquid chromatography (HC-2DLC) to remove major components from herbal extracts, and then characterized the minor ones by mass spectrometry. This strategy was employed to analyze Pueraria lobata and Pueraria thomsonii, the roots of which are used as the Chinese herbal medicine Ge-Gen. Five major compounds in Ge-Gen extract were removed by on-line heart-cutting, and the minor compounds were separated on an RP × RP 2DLC system (1D, Acquity CSH C18, 2.1 × 100 mm, 1.7 μm; 2D, Poroshell Phenyl-Hexyl, 3.0 × 50 mm, 2.7 μm). A synchronized gradient elution program was used to improve chromatographic resolution of the second dimension. By using this 2DLC system, a total of 271 and 254 peaks were separated in P. lobata and P. thomsonii within 35 min, respectively. The practical and effective peak capacity was 1593 and 677, respectively, and the orthogonality was around 70%. Structures of 12 selected compounds were tentatively characterized by mass spectrometry, and 9 of them were discovered from Ge-Gen for the first time. Contents of these minor compounds in Ge-Gen were preliminarily determined to be 0.01-0.1% (w/w). The HC-2DLC/MS system is a powerful and convenient tool to explore minor novel chemical constituents from complex herbal extracts.