Measurement of drug concentrations
Concentrations of CFP and SUL in plasma were simultaneously determined by liquid chromatography method with tandem mass spectrometer detection (LC-MS/MS). Ceftiofur was used as internal standard (IS). The plasma samples were extracted by protein precipitation. An aliquot of 200 µL plasma with 20 µL IS (100 µg mL-1), was added with 400 µL acetonitrile. The mixture was then vortexed for 1 min and centrifuged at 13,500 rpm for 5 min at 4℃. The supernatant was injected into the LC-MS/MS system.
The LC-MS/MS system consisted of an AB ExionLC system and an AB SCIEX QTRAP QUADTM 4500MD (Applied Biosystems Sciex, Ont, Canada). The chromatographic separation was performed using a Waters Symmetry C18 column (150×4.6 mm, 5 µm). The column temperature was set at 40℃. The analysis was carried out using linear gradient elution with mobile phase acetonitrile-0.1% (v/v) formic acid in ammonium formate solution (10 mM), at a flow rate of 1 mL min-1. The linear gradient was as follows: 0-1 min, 10% acetonitrile; 1-7 min, 10% to 90% acetonitrile; 7-8 min, back to the initial state. The total elution time was 8 min. The analytes were detected in negative electrospray ionization mode. Multiple reaction monitoring (MRM) was used to monitor precursor to product ion transition of m/z 644.1→528.1 for cefoperazone, m/z 231.9→140.1 for sulbactam, and m/z521.9→127.0 for IS. Ion spray voltage was at 4500 V and capillary temperature was at 550℃. Declustering potential (DP) was at 30 V for cefoperazone and IS, 12 V for sulbactam. The collision energy (CE) was optimized at 15 eV for cefoperazone, 17 eV for sulbactam, and 30 eV for IS, respectively. Dwell time was set at 100 ms for all the analytes.
The calibration curves ranged from 10 to 500 μg mL-1for cefoperazone and 2 to 100 μg mL-1 for sulbactam, respectively. The accuracy and precision of QC samples were within ±10%.