Gemini 5 μm c18 110a column

As previously described by Tu et al. (2010) (link), preparative HPLC separations were performed on a Gemini 5-μm C18 110A column (30 mm × 50 mm, 5 μm, Phenomenex, Inc., Torrance, CA, United States). A Shimadzu LC-8A binary preparative pump with a Shimadzu SCL-10A VP system controller was connected to the Gilson 215 auto sampler and Gilson 215 fraction collector (Gilson, Inc., Middleton, WI, United States). Detections were performed by a Shimadzu SPD-M20A diode-array detector and a Shimadzu ELSD-LT II evaporative light-scattering detector (Shimadzu Corp., Kyoto, Japan). The mobile phase consisted of water (A) and Acetonitrile (B): 0 min, 98:2; 0.5 min, 98:2; 6.5 min, 0:100; 12.3 min, 0:100; 12.5 min, 98:2; 12.95 min, stop. The flow rate was 25 mL/min. Briefly, fractions (A – fractions 1–6, B – fractions 7–9, C – fractions 10–17, D – fractions 18–31, E – fractions 32–41, F – fractions 42–100) were collected and combined based on mass spectra data; their TLC profiles and biological properties were evaluated (Supplementary Figure S1).

The LC-MS/MS analysis was performed by Agilent 6490 (Agilent Technologies, Santa Clara, CA, USA) coupled with Agilent 1260 HPLC system (Agilent Technologies). The analyte was separated on a Gemini 5 μm C18 110A column (150 × 2 mm i.d., 5 μm, Phenomenex, Torrence, CA, USA) with SecurityGuard Cartridge Kit (Phenomenex). An isocratic mobile phase composed of 0.1% aqueous formic acid and methanol (50:50 v/v %) was used with a flow rate of 0.3 mL/min. The column oven temperature was 40 °C and the total run time was 6 min.
The electrospray ionization (ESI) source was operated in positive mode, and the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms per MRM channel. Gas temperature, gas flow rate, and nebulizer gas pressure were set at 220 °C, 17 L/min, and 45 psi, respectively. The selected precursor/product ion pairs were m/z 174.4 → 165.8 for nafamostat and 177.4 → 168.9 for IS. The fragment voltage was 380 V, and the collision energy was set at 11 eV for both nafamostat and IS. The mass spectrometric data were processed by MassHunter Quantitative Analysis (Agilent Technologies).