Hypersil bds c18

The sample was added into the Hypersil BDS C18 column of a high-performance liquid chromatographic instrument (Shimadzu, Corp., Kyoto, Japan) using a microsyringe. Elution was performed with the mixed solution of methanol, sodium pentanesulfonate and triethylamine as the mobile phase at low temperature at a flow rate of 1 l/min (ultraviolet wavelength, 273 nm; sensitivity, 0.01 AUPS). With deoxycytosine and methyldeoxycytosine standard samples as controls, the DNA methylation content in samples was detected. Each sample was detected 3 times, and the average was taken.

All the reagents were purchased from commercial suppliers (Aldrich®, Shanghai, China; BIDE®, Shanghai, China; or Innochem® Beijing, China), which are used directly without further purification. Chemical HG/T2354-92 silica gel (200–300 mesh, Haiyang®, Qingdao, China) was used for chromatography. ¹H NMR and ¹³C NMR spectra were recorded at room temperature on a Bruker AVANCE III 400 and 500 instrument (Bruker®, Zürich, Switzerland) with tetramethylsilane (TMS) as an internal standard. The following abbreviations are used: s (singlet), d (doublet), dd (two doublets), t (triplet), q (quartet), and m (multiplet). Coupling constants were reported in Hz. High-resolution mass spectra were recorded on a Shimadzu LC‒MS−IT-TOF mass spectrometer (Shimadzu®, Kyoto, Japan). The accurate number of HCl in the molecular structure of 14·3HCl was deduced by an ion chromatography (IC) method with the aid of Dionex ICS-900 (Thermo Fisher Scientific®, Sunnyvale, USA). The purity of compounds was determined by reverse-phase high-performance liquid chromatography (HPLC) analysis confirming to be over 95%. HPLC instrument: Shimadzu LC-20AT (Shimadzu®, column: Hypersil BDS C18, 5.0 μm, 150 mm × 4.6 mm (elite); detector: SPD-20A UV–Vis detector, UV detection at 254 nm; elution, methanol/H₂O (100%–90%, v/v); column temperature: 25 °C; and flow rate = 0.8–1.0 mL/min.