Use of on-line stop-flow heart-cutting two-dimensional high performance liquid chromatography for simultaneous determination of 12 major constituents in tartary buckwheat (Fagopyrum tataricum Gaertn)

• An on-line stop-flow heart-cutting 2D-LC was developed.
• It offered different separation selectivity and high chromatographic resolution.
• 2D-LC provided a way for resolving similar structures but different polarities.
• Fractionation was achieved in the combination of back-flush and direct-flow.

The use of two-dimensional liquid chromatography (2D-LC) for quantification studies presents challenges with respect to repeatability, precision, and robustness. The present study used an on-line stop-flow heart-cutting 2D-LC system to determine 12 chemical constituents in tartary buckwheat. A combination of various stationary phases was developed and bridged using two switch valves as the interface. Hydrophilic interaction chromatography was chosen for separation in the first dimension (1D), and mixed mode stationary phases (an amide polar-embedded phase and alkyl-phenyl phase) were used in parallel for separation in the second dimension (2D). The mobile phase comprised acetonitrile and water containing 0.03% aqueous phosphoric acid. The sample was separated into two fractions on the 1D column (HILIC-10 column) using 5% acetonitrile. One fraction, mainly comprising flavonoids, was directly eluted onto the head of 2D column (Polar Advantage II column) and further separated using a linear gradient of 11–23% acetonitrile. The second fraction, containing phenylpropanoid glycosides, was trapped on the 1D column. This retained fraction was back-flushed onto the 2D column (Phenyl-1 column) and separated using a linear gradient of 35–43% acetonitrile. An on-line stop-flow heart-cutting 2D-LC system was successfully developed with column switching and back-flush. This 2D-LC system was validated and was able to simultaneously determine 12 major components in tartary buckwheat: seven flavonoids, four phenylpropanoid glycosides, and N-trans-feruloyltyramine. The system showed good performance with respect to linearity (r > 0.996), repeatability (RSD, relative standard deviation < 3.4%), intra-day and inter-day precision (RSD < 4.6%), recovery (91.2–108%), limit of detection (LOD) (0.05–0.21 μg/mL), and limit of quantification (LOQ) (0.10–0.41 μg/mL). The on-line stop-flow heart-cutting 2D-LC system offers a potential approach to analyze compounds, which have similar structures but different polarities, in herbal medicines.