Establish a simple LC-MS/MS method for the determination of MRX-8 by employing dried blood spots in rat pharmacokinetic study.

The rising prevalence of extensively drug-resistant gram-negative infections necessitates safer polymyxin analogs, as conventional polymyxins are limited by nephrotoxicity and compositional heterogeneity. MRX-8, a next-generation polymyxin B1 derivative engineered to reduce toxicity while maintaining potent antibacterial activity (MIC90 <1 mg/L), demonstrates improved pharmacokinetic/pharmacodynamic (PK/PD) targets. To overcome challenges in preclinical blood sampling volume, we developed a LC-MS/MS method to quantify MRX-8 in dried blood spots (DBS). DBS samples (40 μL blood) collected on Whatman 903® cards were extracted with formic acid-water-acetonitrile (6:64:30, v/v/v) and analyzed using an AB Sciex QTRAP 5500/Shimadzu LC-30A system with MRM transitions at 617.5→155.1 (MRX-8) and 402.3→101.2 (polymyxin B1 internal standard). The method demonstrated linearity (0.0200-10.0 mg/L), precision (3.6-14.1 %), accuracy (87.4-107.8 %), recovery (90.1-102.2 %), and matrix effects (108.1-125.4 %). DBS stability was confirmed under room temperature (3 days), refrigerated (2-8 °C, 7 days), and frozen (-20 °C/-70 °C, 25 days) conditions. Adjusted DBS concentrations incorporating a plasma-blood distribution coefficient (0.59) correlated with plasma pharmacokinetics (R² = 0.976). Compared to conventional plasma sampling, this DBS approach reduces blood volumes and significantly minimizes animal burden. This validated microsampling method provides an ethical and efficient solution for preclinical PK evaluation of MRX-8, accelerating its development as a critical antibacterial therapy against drug-resistant pathogens.