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%.