Simultaneous Quantification of Colistin A and B in Human Plasma Using a Small Volume with a High-Throughput LC-MS/MS Method

Background/Objectives: Colistin is a complex polymyxin antibiotic with a narrow therapeutic window and significant interindividual pharmacokinetic variability, necessitating precise concentration monitoring. Current analytical methods often utilize colistin mixtures or require large sample volumes, potentially limiting the precision and resolution of individual component quantification. This study aimed to develop a sensitive and component-specific bioanalytical assay for the simultaneous quantification of colistin A and colistin B in human plasma. Methods: A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed using pure, component-specific reference standards to ensure rigorous independent quantification of each component. Analytes were efficiently extracted from a small volume of plasma (50 µL) using solid-phase extraction. Chromatographic separation was achieved on a C18 column with a total runtime of 4 min, and detection was performed using negative-ion multiple reaction monitoring (MRM). Results: Calibration curves showed excellent linearity over a range of 0.1–20 µg/mL for both colistin A and B (R2 > 0.99). The precision (%CV ≤ 8.8%) and accuracy (86.4–105.7%) for both components met the predefined regulatory criteria. This method was clinically validated using 60 plasma samples from 15 patients, demonstrating its applicability for capturing individual concentration–time profiles within the clinically relevant range (0.323–19.579 µg/mL for colistin A and 0.065–6.132 µg/mL for colistin B). Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a high-throughput workflow using a small plasma volume. Therefore, it serves as a practical tool for therapeutic drug monitoring (TDM)-guided dose optimization and further clinical investigations of colistin therapy.