High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
The concentration of antibiotics in each plasma sample was measured using a validated LC-MS/MS method. Detailed measurement conditions are summarised in Table S1. The protein binding rates of nacubactam and aztreonam in mouse plasma were determined by the ultrafiltration method. In short, plasma containing nacubactam and aztreonam at concentrations of 1, 10, and 400 mg L-1 (n = 3 per concentration) was incubated at 37 °C for 30 min and then set into Centrifree® 4104 Ultrafiltration Centrifugal Filters (Millipore, Bedford, MA, USA). The filtrate was obtained by centrifugation at 1,500 g for 5 min. The concentrations of the antibiotics in the initial sample and the filtrate were measured using LC-MS/MS, and the protein binding rate was calculated.