In detail, 10 mg of HNCP3 and UNCP4 were dissolved in 1.5 mL of DMSO (1.5 mL), then 1.5 mL of NaOH (50%)-DMSO solution (V:V = 1:1) was added and reacted at 30 °C for 3 h. Methyliodide (0.5 mL) was added and stirred at 30 °C for 2.5 h in dark place under N2 protection, and the reaction was terminated with 1 mL of deionized water. The above operation step was repeated three times until the absorption peak of OH group disappeared substantially. The methylated HNCP3 and UNCP4 were further hydrolyzed with H2SO4 (2 mol/L) at 120 °C for 2 h and dried by rotary evaporation under the protection of nitrogen. The rotary-dried sample was dissolved with NaOH solution, and shaken with the adding of 25 mg of sodium borohydride at 25 °C for 2 h, the pH was adjusted to 5.5–7.0 with acetic acid for removing excess sodium borohydride.
Subsequently, the reduced samples were acetylated by mixing with 0.7 mL of pyridine and 1 mL of acetic anhydride at 90 °C for 30 min. The dried reaction product was dissolved in ethyl acetate and then analyzed by a GLC-MS system (THERMO 1310 GC-ISQ LT MS, USA) apparatus equipped with a TG-200MS capillary column (30 mm × 0.25 mm, 0.25 µm). The specific program was as follows: from 160 to 210 °C, the temperature was raised at a rate of 2 °C/min, then increased to 240 °C at a rate of 5 °C/min, and finally kept for 20 min. MS scan range was set at m/z 35–4000.
Subsequently, the reduced samples were acetylated by mixing with 0.7 mL of pyridine and 1 mL of acetic anhydride at 90 °C for 30 min. The dried reaction product was dissolved in ethyl acetate and then analyzed by a GLC-MS system (THERMO 1310 GC-ISQ LT MS, USA) apparatus equipped with a TG-200MS capillary column (30 mm × 0.25 mm, 0.25 µm). The specific program was as follows: from 160 to 210 °C, the temperature was raised at a rate of 2 °C/min, then increased to 240 °C at a rate of 5 °C/min, and finally kept for 20 min. MS scan range was set at m/z 35–4000.