*2.4. D-LC Analysis*

The 2D-LC system was composed of an ACK 3200 column oven from ANAX with a fully automatic two-dimensional chromatographic coupling instrument (Changsha, China) and an LC-20AT high-performance liquid chromatograph from Shimadzu (Kyoto, Japan), which included a high-pressure LC-20AT pump (PUMP-B), two low-pressure gradient chromatography LC-20AT pumps (PUMP-A and PUMP-C), a CBM-20A system controller, a SIL-20A autosampler equipped with a 1000 μL injection loop and an SPD-20A UV detector.

The 2D-LC system used a heart-cutting transfer mode between the LC1 column and the LC2 column. The samples were first separated in 1D LC (LC1 column) mode to eliminate the interference of most impurities. The second step is the MC mode, in which the target material is transferred to the intermediate column after heart-cutting, where it is enriched. In the third step, the target was transferred to 2D LC (LC2 column) for further separation and, finally, UV detection. The mode switching in the whole process is done by LabSolutions LC Workstation V ER.5. The workstation corresponding to each mode can be highly automated to complete the corresponding work.

The first-dimensional (1D) liquid chromatography separation column was a strong cation exchange column (ASTON SXI 3.5 × 25 mm i.d.; particle size, 5 μm, Changsha, China). The middle column was a bonded phenyl column (SN-MA 4.6 × 10 mm i.d.; particle size, 5 μm, Changsha, China). The second-dimensional (2D) chromatography column was a reversed-phase column (SBR-2A 4.6 × 10 mm i.d.; particle size, 5 μm, Changsha, China). The mobile phase of PUMP-A was a 27:10:63 (*v*/*v*/*v*) solution of 80:20 (*v*/*v*) acetonitrile/water-10 mmol/L ammonium dihydrogen phosphate buffer (pH = 7.5)–10 mmol/L ammonium dihydrogen phosphate buffer (pH = 3.0). The PUMP-B mobile phase was water. The PUMP-C mobile phase was a 35:46:16.4:1.6:1 (*v*/*v*/*v*/*v*/*v*) solution of water–acetonitrile–methanol–85% phosphoric acid–acetic acid (pH = 7.0). The temperature of the column was maintained at 40 ◦C, and the injection volume was 500 μL. The flow rate of PUMP-A is 1.2 mL/min; the PUMP-C flow rate is 1 mL/min. The detector wavelengths were 254 nm and 263 nm.
