Lc 20ad solvent

Samples were analysed using a liquid chromatographic system (Shimadzu, Kyoto, Japan) equipped with a CBM-20A system controller connected to an LC-20AD solvent delivery unit, DGU-20A3 degasser system, CTO-10ASVP column oven, SIL-20AHT autosampler and SPD-20A ultraviolet-visible detector. The isocratic mobile phase used was methanol and 0.1% of trifluoroacetic acid in distilled water at a volume ratio of 85:15 and a flow rate of 1 mL/min. The mobile phase was filtered through a 0.45 µm nylon membrane filter under a vacuum condition and sonicated before use. A Phenomenex^® Luna C18 column (250 × 4.6 mm ID, 5 μm; Torrance, CA, USA) fitted with a Thermo Scientific Uniguard™ guard column (Waltham, Boston, MA, USA) with an ODS C18 cartridge (10 × 4 mm ID, 5 μm) was used as stationary phase. UV detection was set at 305 nm and a sample injection volume of 40 μL was used with a column temperature of 40 °C. An internal standard of MS was used during method development and validation. The retention time of MS was ~4.6 min. The limits of detection and quantification for MS were 0.08 and 0.25 µg/mL for a low drug concentration range of 0.25–7 µg/mL, respectively, as well as 4.20 and 12.72 µg/mL for a high drug concentration range of 5–150 µg/mL, respectively.