2.5. Reverse−Phase High−Performance Liquid Chromatography (RP−HPLC) Purification

To establish the purification conditions, the elution fractions were loaded onto a Wukong K2025 RP−HPLC apparatus (Haineng Future Technology Group Co., Ltd., Shanghai, China) with a column of SinoChrom 300Å ODS−AP C18 (4.6 mm × 250 mm × 5 μm, Dalian Yilite Analytical Instrument Co., Ltd., Dalian, China). The established chromatographic conditions were as follows: The mobile phase was acetonitrile solution containing 1% TFA and 1% TFA, with an injection volume of 90 μL, an elution time of 25 min, a flow rate of 1 mL/min, and a detection wavelength of 220 nm. For gradient elution, 1% TFA solution and deionized water were maintained in a volume ratio of 98:2 for 5 min, in a volume ratio of 50:50 for 5 min, in a volume ratio of 10:90 for 10 min, and finally, in a volume ratio of 98:2 for 5 min.

According to the established chromatographic conditions, 5′−NT was prepared using an Agilent 1260 preparative liquid chromatography apparatus equipped with diode array detectors (Agilent Technology (China) Co., Ltd., Beijing, China) and a column of SinoChrom 300Å ODS−AP C18 (10 mm × 250 mm × 5 μm, Dalian Yilite Analytical Instrument Co., Ltd., Dalian, China). A sample of 425 μL in volume was loaded and separated at a flow rate of 4.7 mL/min to obtain purified 5′−NT. A diode array detector was used for full−band scanning during elution, and the other parameters were the same as those of the analysis conditions above.