2.4 Blood sampling and analytical method
Blood sample collection
The patients were divided into two groups. The first group contained 42 patients. Blood samples were collected from the first group at 5 h and 7.5 h after the sixth dosing. A total of 84 concentration data points were collected. The other group contained 12 patients. Blood samples were obtained at 0, 0.5, 1, 2, 4, 6, 8, and 12 h after the sixth dosing. A total of 96 concentration data points were collected from the second group.
Imipenem analysis method
All blood samples were placed into EDTA-coated tubes. The plasma and blood cells were separated within 2 h. Samples were centrifuged at 3000 rpm for 10 min at 4°C and stored at -80°C until further use. A selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the simultaneous quantification of imipenem in human plasma. Liquid–liquid extraction using 400 µL of acetonitrile was used to extract imipenem and the internal standard (IS; Meropenem-d6) from 200 µL of human plasma. The analytes were chromatographically separated using an Acquity UPLC BEH C18 column (50×2.1 mm, 1.8 μm) with 0.1% FA in acetonitrile and 2 mM ammonium acetate in water. The mobile phase was produced with a composition of 50% water to 50% acetonitrile containing 0.1% FA and delivered at a flow rate of 0.2 mL/min. The total run time was 6 min. Mass detection was performed using a Waters Xevo TQ-S triple-quadrupole mass spectrometer in positive electrospray ionization mode. The mass transitions selected were m/z 300.0→171.0 and m/z 390.1→147.0 to quantify RST and IS, respectively.