Gemini nx 5u c18 110a column

The HPLC method used to determine the coumarin content of the different Kampo medicines was modified from that used for the determination of the (E)-cinnamic acid contained in keishibukuryogan extract found in the Japanese Pharmacopoeia 16th Edition (Ministry of Health, Labour and Welfare, Japan, 2011 ). A Shimadzu LC10A system (Shimadzu Co., Kyoto, Japan) equipped with a photodiode array detector (SPD-M10A_{V P}) was used. A Gemini-Nx5u C18 110A column (250 mm × 4.6 mm I.D., 5 μm, Phenomenex, Torrance, CA, USA) was used with a mobile phase that consisted of a mixture of water, acetonitrile, and trifluoroacetic acid (750:250:0.5, v/v/v) delivered at a flow rate of 0.5 mL/min. The column temperature was maintained at 40°C. The absorbance detector was set at 273 nm.

The peptide mixture was resolved in a 20-mM ammonium formate buffer A (20-mM ammonium formate in pure water, pH 10.0) and fractionated by high-pH reverse-phase separation using LC-20AB HPLC system (Shimadzu, Japan) with a 4.6 mm × 150 mm, Gemini-NX 5u C18 110A column (Phenomenex, Guangzhou, China). High-pH reverse-phase separation was performed using a linear gradient starting from 5% buffer B to 80% buffer B in 30 min (buffer B: 20 mM ammonium formate in 100% acetonitrile, pH 10.0). Sixteen fractions were collected and dried in a vacuum concentrator rotation vacuum (Christ RVC 2-25, Christ, Germany). Fifty microliter buffer C (0.1% formic acid in 5% acetonitrile) (TEDIA, Fairfield, USA) was added to each dried fraction for RPLC-MS/MS (reversed-phase liquid chromatography-mass spectrum/mass spectrum) analysis.