Eclipse sb c18 column

For plasma pharmacokinetic and tissue distribution studies, the LC-MS/MS system consisted of a Shimadzu LC-20A HPLC system and an Applied Biosystems Sciex API 4000⁺ (MDS-Sciex, Concord, Canada) equipped with a Turbo Ion Spray. Data acquisition and processing were performed using Analyst 1.6.3 software (Applied Biosystems, Foster City, CA, USA). Chromatographic separation was carried out on an Agilent Eclipse XDB-C₁₈ (2.1 × 150 mm, 5 μm), and the column temperature was maintained at 40°C. The mobile phase was composed of acetonitrile-water containing 0.1% formic acid (32 : 68, v/v). The flow rate was set at 0.3 mL/min. The autosampler was set at 4°C and the injection volume was 5 μL.
In the study on metabolism of icariin, metabolites were identified by LTQ-Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany) coupled with an ESI source. An Agilent Eclipse SB-C₁₈ column (2.1 × 100 mm, 1.8 μm) was employed and column temperature was maintained at 40°C. Mobile phase was composed of A (0.1% formic acid aqueous solution) and B (acetonitrile) with a gradient elution as follows: 0–35 min, 15–30% B; 35–55 min, 30–50% B; 55–70 min, 50% B; 70–80 min, 50–100% B; 80–90 min, 100% B. The flow rate of mobile phase was 0.2 mL/min. The injection volume of samples was 5 μL.

For fermentation, strain NRRL2564 and its mutants were grown in the seed medium TSBY for 24 h at 30 °C; then, 4% inoculum was transferred into the fermentation medium GSY (15 g/L glucose, 5 g/L soluble starch, 5 g/L yeast extract, 5 g/L NaCl, 5 g/L CaCO₃, pH 7.2) and cultured for 4 days at 220 rpm, 30 °C.
To detection the production of mitomycin C in the wild-type strain NRRL2564, the mutants, and the complemented recombinants, 50 mL of fermentation broth was extracted with 50 mL of ethyl acetate. After vacuum evaporated, the crude extract was dissolved in 1 mL methanol and passed through 0.22 μm filters, and prepared for HPLC (Agilent series 1260, Agilent Technologies, Santa Clara, CA, USA) analysis. The HPLC was monitored under the following conditions: column, Agilent Eclipse SB-C18 column (250 × 4.6 mm, 5 μm); mobile phase, 0.1% formic acid in water (solvent A) and acetonitrile (solvent B); flow rate: 0.6 mL/min; gradient condition, 85% A: 15% B for 5 min, 80% A: 20% B at 6 min, 40% A: 60% B at 30 min, 10% A: 90% B at 31 min, and after 5 min, the conditions returned back to 85% A: 15% B for 5 min; injection volume, 10 μL; and column temperature, 37 °C, UV 363 nm.