Agilent eclipse xdb c18 column

Optical rotation spectra were recorded on a Modular polarimeter MCP 5100 (JASCO, Tokyo, Japan). ECD spectra and UV spectra were measured using a Biologic MOS450-SFM300 instrument (JASCO, Tokyo, Japan). The Spartan 14 program (Wavefunction Inc., Irvine, CA, USA) was used for calculating Merck molecular force field (MMFF). The Gaussian 16 program package1 was used for density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations. NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for ¹H NMR and 100 MHz for ¹³C NMR) (Bruker Corporation, Switzerland) instrument using DMSO-d₆ as a solvent. Tetramethyl silane (TMS) was used as an internal standard. HRESIMS spectrometer were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. (Billerica, MA, USA). Semi-Preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDBC18 column (9.4 250 mm, 5 µm) (Agilent Corporation, Santa Clara, CA, USA). Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200–300 mesh) and octadecyl silyl silica gel (YMC; 12 nm–50 µm) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co. (Yantai, China); G60, F-254) were used for thin-layer chromatography (TLC).

Optical rotations were measured with a JASCO P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were recorded on a Beckman DU 640 spectrophotometer (Beckman Coulter, Inc., Miami, USA). IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer (Thermo Fisher Scientific Inc., Massachusetts, USA) in KBr discs. HR-ESI-MS spectra were measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer (waters corporation, Milford, USA). NMR spectra were recorded on Bruker 400 MHz spectrometers (Bruker Daltonics Inc., Karlsruhe, Germany) using TMS (Tetramethyl silane) as an internal standard δ. X-ray diffraction data were collected on an Agilent Technologies Gemini A Ultra system (Agilent Technologies Inc., Palo Alto, USA). Semipreparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C₁₈ column (250 × 10.0mm, 5 µm). Silica gel (300-400 mesh, Qingdao Marine Chemical Inc., China), Silica gel (200-300) mesh (Qingdao Marine Chemical Inc., China), Silica gel H (10-40 μm, Qingdao Marine Chemical Inc., China), Lichroprep RP-18 gel (40-63 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (40-70 μm, Amersham Biosciences, Sweden) were used for column chromatography (CC).

All samples were analyzed using a Shimadzu LC-20AD HPLC system equipped with an SPD-M20A detector and LC solution software (Shimadzu Corporation, Kyoto, Japan). An Agilent Eclipse XDB C18 column (150 × 4.6 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA) was used for the analysis, and the column temperature was set at 35 °C. Furosemide samples were dissolved in a solvent mixture (acetonitrile/water/glacial acetic acid = 500:500:0.1, v/v/v). The buffer solution was prepared by dissolving 1.36 g of KH₂PO₄ in 1000 mL of water and the pH was adjusted to 3.0 with phosphoric acid. The mixture of buffer solution and methanol (90/10, v/v) was used as mobile phase A, and mobile phase B was a mixture of buffer solution and methanol (50/50, v/v). The LC gradient program was set as follows: time (min)/% B: 0/10, 10/63, 20/63, 30/100, 45/100, 55/10, and 60/10. The injection volume was 10 μL. The flow rate was 0.8 mL/min with a detection wavelength of 230 nm.