The stock standard solutions (1000 μg mL−1) were prepared individually for the selected seven drug standards by weighing 10 mg of reference standard substances and dissolving them in 1:1 mL of methanol:deionized water (v/v) in 10 mL volumetric flask. The stock solutions were stored at −18 °C and prepared fresh every 1 month. Mixed stock solutions of the seven antibiotic standards (200 μg mL−1) and the series of working standard solutions for the method development were prepared daily with a mobile phase dilution.
All the standard solutions prepared for the LC were filtered through a 0.45 μm nylon syringe membrane filter before use. The mobile phase was filtered through the Millipore glass filter (Millipore filter cellulose nitrate gridded with 0.22 μ size and 47 mm diameter) assembly attached with vacuum pump and was sonicated with ultrasonic machine, B5510J-DTH (Branson, Danbury, CT), for 30 min before pumping into HPLC system for degassing.
All the standard solutions prepared for the LC were filtered through a 0.45 μm nylon syringe membrane filter before use. The mobile phase was filtered through the Millipore glass filter (Millipore filter cellulose nitrate gridded with 0.22 μ size and 47 mm diameter) assembly attached with vacuum pump and was sonicated with ultrasonic machine, B5510J-DTH (Branson, Danbury, CT), for 30 min before pumping into HPLC system for degassing.