Analytical evaluation for the quantitation of fifteen antibiotics in plasma by liquid chromatography coupled to high-resolution, accurate-mass measurements for clinical research

Significance of the Topic

Accurate quantification of multiple antibiotics in plasma is essential for clinical pharmacokinetics and therapeutic drug monitoring. Traditional HPLC-UV methods often require extensive sample preparation and target only a limited number of compounds. High-resolution, accurate-mass (HRAM) LC-MS enables simultaneous analysis of diverse antibiotic classes with improved specificity and streamlined workflows.

Objectives and Overview of the Study

This work aimed to develop and analytically evaluate a rapid method for quantifying fifteen antibiotics spanning penicillins, cephalosporins, carbapenems, quinolones, lincosamides, and other β-lactamase inhibitors in human plasma. The method uses a Q Exactive Focus quadrupole-Orbitrap mass spectrometer coupled to a UHPLC system.

Methodology and Instrumentation

• Sample preparation: Protein precipitation with methanol/formic acid followed by 1:4 dilution; 5 μL injection volume.
• Chromatography: Thermo Scientific UltiMate 3000RS UHPLC; Accucore C18 column (100×2.1 mm, 2.6 μm) at 40 °C; gradient elution from 2% to 100% organic in 9 minutes; 0.5 mL/min flow.
• Mass spectrometry: Q Exactive Focus quadrupole-Orbitrap with HESI-II probe in positive mode; full scan m/z 120–650 at 35,000 resolution; 5 ppm extraction window.
• Analytes: Fifteen antibiotics with seven isotopically labeled internal standards.
• Calibration and validation: Seven calibration levels (0.5–32 mg/L); QC levels at 0.5, 1.5, 4, and 25 mg/L; LODs between 0.02 and 0.10 mg/L; LOQ set at 0.5 mg/L.

Main Results and Discussion

The method demonstrated linear responses across the calibration range for all compounds. Intra-day and inter-day precision (%CV) remained below 15% for QC levels above LOQ and below 20% at the LOQ. Accuracy was within ±15% for most analytes and within ±20% at the LOQ. Representative extracted ion chromatograms at the LOQ showed clear peak shapes and retention times. Imipenem exhibited tautomer-related peak splitting, effectively resolved under the chosen LC conditions.

Benefits and Practical Applications

This approach enables simultaneous quantitation of multiple antibiotic classes in a single 9-minute run with minimal sample preparation. High mass accuracy ensures high specificity, making the method suitable for clinical research, therapeutic monitoring, and pharmacokinetic studies.

Future Trends and Applications

Expanding the panel to include additional antibiotics and metabolites, integrating automated sample handling, and applying the workflow to high-throughput clinical settings represent key future directions. Coupling HRAM LC-MS with data-driven decision tools could further enhance personalized antibiotic dosing.

Conclusion

A robust, rapid HRAM LC-MS method was developed for the quantification of fifteen antibiotics in plasma. The workflow offers adequate sensitivity, precision, and accuracy with straightforward sample preparation, supporting various clinical research applications.