Thermo hypersil gold c18 column

An HPLC separation module (Thermo Scientific UltiMate 3000) with a cooled autosampler and a column oven was used for this assay. A Thermo Hypersil GOLD C18 column (50 mm × 2.1 mm, 1.9 µm; Thermo Fisher) was used for chromatographic separation. The gradient elution was performed as follows:
Simvastatin and simvastatin hydroxy acid: injection volume, 4 μL; internal standard, lovastatin and lovastatin hydroxy acid; flow rate, 0.3 mL/min; column temperature, 35 °C; Linear gradient elution procedure is shown in Table 9.
Sildenafil and N-desmethyl sildenafil: injection volume, 3 μL; internal standard, diazepam; flow rate, 0.3 mL/min; column temperature, 35 °C; Linear gradient elution procedure is shown in Table 10.
Nifedipine and dehydronifedipine: injection volume, 8 μL; internal standard, nimodipine; flow rate, 0.3 mL/min; column temperature, 35 °C; Linear gradient elution procedure is shown in Table 11.
Bosentan and hydroxybosentan: injection volume, 8 μL; internal standard, losartan; flow rate, 0.3 mL/min; column temperature, 35 °C; Linear gradient elution procedure is shown in Table 12.

A Shimadzu Prominence UPLC (Shimadzu, United States) system coupled to an AB SCIEX™ 5500 Q-Trap® mass spectrometer (Applied Bio-systems, United States) equipped with an electrospray ionization interface operated in negative multiple reaction monitoring (MRM) mode were applied to analysis. The analytes separation was achieved via gradient elution of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.5 mL/min on a Thermo Hypersil Gold C₁₈ column (50 mm × 2.1 mm, 3 μm; Thermo Scientific, New York, United States). The run time was 4.0 min for each analysis. The gradient elution program was used as follows: 5% B→10% B at 0–0.2 min, 10% B→70% B at 0.2–2.0 min, 70% B at 2.0–2.5 min, 70% B→5% B at 2.50–2.51 min; 5% B at 2.51–4.0 min. The temperatures of autosampler and column were set at 15°C and 40°C, respectively. The supernatant injection volume was 2 μl. The MRM conditions (source-dependent mass parameters) were defined as follows: Ion Spray Voltage, −4500 V; temperature, 550°C; curtain gas, 35.0 psi; collision gas, medium; Gas 1, 55.0 psi; Gas 2, 55.0 psi. The transitions of m/z 300.9→107.1, m/z 419.3→297.1, m/z 160.9→77.0, m/z 567.1→243.2 and m/z 283.1→268.2 were selected for morin, morusin, umbelliferone, mulberroside A and IS, respectively. The system control and data analysis were performed using AB SCIEX Analyst software (version 1.6.3).

Chromatographic analysis was carried out employing an Ultimate 3000 HPLC system equipped with an Ultraviolet–visible spectroscopy (UV-Vis) diode-array detector (DAD) (Thermo Fisher Scientific, Waltham, MA, USA). Separation was performed by using a Thermo hypersil gold C18 column (250 × 4.6 mm i.d., 5 μm particle size, Thermo Fisher Scientific). The mobile phases were prepared from 0.1% (v/v) formic acid in water (eluent A) and acetonitrile (eluent B). The gradient was as follows: 0–40 min, linear gradient from 5% to 80% B. Then, the gradient returned to 5% of eluent B and this composition was held for 10 min for re-equilibrate the column. The column temperature was set at 25 °C, the injection volume was set at 10 μL, and the flow rate was 1 mL/min. Gigantol was monitored by DAD and quantified at 279 nm. Calibration curves were calculated for gigantol on the basis of six different concentrations from 0.625 to 20 μg/mL. The calibration curve was Y = 3.7649X + 0.0481, R² = 1, where y is the concentration (μg/mL) and x is the peak area. This formula is suitable for linearity in the range of 0.625 μg/mL to 20 μg/mL.