Dominant doubly charged ion enabling robust LC-MS/MS quantification of a novel small-molecule antibacterial drug Cefidelbaz in human plasma and its application to pharmacokinetic and protein binding studies.

Cefidelbaz is a novel siderophore cephalosporin antibacterial agent with a unique "Trojan horse" mechanism, which can combat some of the most difficult-to-treat Gram-negative bacterial infections. In mass spectrometric analysis, cefidelbaz generated distinct precursor ions in both positive and negative modes during the first-quadrupole full-scan (Q1 full-scan), while multiple precursor ions with different charge states were observed in positive mode. Notably, singly charged precursor ions in either positive or negative mode failed to provide wide linear range and high sensitivity, whereas the doubly charged precursor ions enabled reliable and reproducible quantitative analysis. These findings, together with reported charge-state behavior of cephalosporins with zwitterionic character, demonstrated that the optimal precursor ions should be selected according to experimental quantitative performance. Here, two independent LC-MS/MS (liquid chromatography-tandem mass spectrometry) methods were developed. The first method was established for the quantification of total cefidelbaz in human plasma, with a calibration range of 1.00-600 µg/mL. The second method, based on ultrafiltration coupled with LC-MS/MS, was developed for the determination of plasma protein binding (%PPB), covering a calibration range of 0.500-250 µg/mL. Overall, this study demonstrated the importance of precursor ions selection for reliable LC-MS/MS quantification of cephalosporins with zwitterionic character and provides validated bioanalytical methods to support clinical development of cefidelbaz.