Rp18 guard column

A liquid chromatography system consisting of an HP 1200 Series (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, degasser system, automatic injector and column thermostat, and an Agilent 6140 single quadrupole mass spectrometer (Agilent Technologies) was used for the analysis. Separations were performed on a reverse-phase Kinetex C18 column (100 mm × 4,6 mm; 2,6 μm) and an RP18 guard column (4.0 mm × 3.0 mm, 5 μm), both from Phenomenex (Torrance, CA, USA). The column thermostat temperature was set to 20°C. The flow rate was 0.5 mL/min and injection volume 10 μL. The composition of mobile phases A and B was set as 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution started from 10% of solvent B at 0 min, increased to 20% from 4.01 to 8.0 min, to 40% from 8.01 to 12.0 min, to 50% from 12.01 to 15.0 min, reached 100% from 15.01 to 17.0 min, and was held for 1 min; next reduced to 10% from 18.01 to 19.0 min and was held from 19.01 min to 23 min which was the end of the analysis run time. Electrospray ionisation (ESI) was set in a positive mode, the capillary voltage was set at 2,000 V, drying gas temperature was 350°C, drying gas flow was 12 L/min, and nebulising gas pressure was 40 psi. Selected ion monitoring and retention time for all antibiotic substances intended for detection are listed in Table 2.