Symmetry c18 guard column

Erlotinib levels in homogenized tumor tissues were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV detection at 345 nm. Separation was achieved on a Waters Symmetry C18 column (150 × 4.6 mm, 5.0 μm; Waters, Milford, MA) preceded by the use of a Symmetry C18 Guard column (3.9 × 20 mm). The mobile phase was 50 mM potassium phosphate buffer (pH 4.8) containing 0.2 % triethylamine and acetonitrile (60:40, v/v), with 1.0 mL/min flow rate at 25 °C. Sample pretreatment involved mixing 500 μL of tumor tissue homogenate with 80 μL of internal standard (70 μg/mL of midazolam in methanol) and 5 mL of tert-butyl methyl ether for 10 min. After centrifugation (650 g, 10 min, 4 °C), the organic top layer was transferred to a clean tube and dried under nitrogen gas at 37 °C. The residue was dissolved in 250 μL of mobile phase. The solution was centrifuged (4,000 g, 30 min) and the supernatant was passed through a microporous membrane filter (Millex-GV 0.22-μm filters, Millipore Corp., Bedford, MA). Insoluble materials were removed by filtration, and the filtrate was analyzed by high-performance liquid chromatography. The calibration curves were linear over a concentration range of 20–4,000 ng/mL (r2 > 0.998).

The method was developed with a Nexera X2 UHPLC system comprising two LC-30AD pumps with degassers, SIL-30AC autosampler, CTO-30AD oven and SPD-M30A photo diode-array detector, controlled by LabSolutions software (Shimadzu Corp., Kyoto, Japan).
The stationary phase was a Symmetry C18 (2.1 × 100 mm, 3.5 µm) analytical column (Waters, Milford, USA) preceded by a Symmetry C18 guard column (2.1 × 10 mm, 3.5 µm) (Waters). Mobile phase was 20 mM sodium phosphate buffer at pH 3.0 in 12.5% acetonitrile delivered isocratically at 0.25 mL/min. The autosampler was held at 4°C, and injection volume was 0.5 µL. The photodiode-array detector scanned from 200 to 500 nm and ceftazidime, avibactam and pyridine were quantified at wavelengths of 260, 230 and 254 nm, respectively.