Automated HILiC-MS/MS Method for Therapeutic Drug Monitoring of Aminoglycoside Antibiotics and Vancomycin

Importance of the Topic

Aminoglycoside antibiotics and vancomycin are critical agents for treating severe infections, particularly those caused by Gram-negative and resistant Gram-positive bacteria. Their narrow therapeutic windows and nephrotoxic potential make precise therapeutic drug monitoring essential. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) offers superior selectivity, multiplexing capability, and lower per-sample cost compared to traditional immunoassays.

Objectives and Study Overview

This study aimed to develop and validate a fully automated hydrophilic interaction liquid chromatography (HILIC) coupled with MS/MS method for simultaneous quantification of seven aminoglycosides and vancomycin in plasma. Key goals included achieving fast analysis, high accuracy, and robust throughput suitable for routine clinical laboratory workflows.

Instrumentation Used

• Nexera X2 UHPLC system for chromatographic separation on an amide HILIC column (GL Sciences InertSustain Amide, 3 µm, 50 × 2.1 mm).
• LCMS-8060 triple quadrupole mass spectrometer with heated ESI source (positive mode at +5 kV).
• CLAM™-2000 automated sample preparation device.

Methodology

• Reagents: Analytical standards of amikacin, gentamicin isomers, kanamycin, neomycin B, paromomycin, tobramycin, vancomycin, and hygromycin B (internal standard).
• Calibration: Seven concentration levels (0.1–50 µg/mL; vancomycin up to 100 µg/mL; arbekacin 0.1–30 µg/mL) prepared in blank plasma.
• Sample Preparation: Automated precipitation with trichloroacetic acid, filtration, and direct transfer to the autosampler for analysis.
• Chromatography: Gradient elution from 75% to 55% acetonitrile in 1.3 min at 400 µL/min; total run time 4.75 min.
• MS/MS Detection: Multiple reaction monitoring transitions optimized for each compound; dwell time 23 ms.

Results and Discussion

• Calibration: Quadratic regression with 1/x or 1/x² weighting yielded excellent linearity (R² > 0.997) across the calibration ranges.
• Recovery and Matrix Effects: Total recoveries between 89% and 105%, with relative standard deviations (RSD) below 12%.
• Precision and Accuracy: Intra- and inter-day RSD < 15% (20% at lower limit), accuracy within 80–120% across four QC levels.
• Throughput: Automated sample preparation overlapped with analysis, enabling rapid processing and consistent performance.

Benefits and Practical Applications of the Method

• High selectivity and minimal cross-reactivity compared to immunoassays.
• Simultaneous quantification of multiple antibiotics in a single run.
• Short analysis time and automated workflow improve laboratory efficiency and reduce hands-on time.
• Suitable for routine therapeutic drug monitoring in clinical and pharmaceutical quality control settings.

Future Trends and Possibilities for Application

Integration of this automated HILIC-MS/MS approach with advanced laboratory information systems can further streamline clinical workflows. Expanding the panel to include additional antimicrobial agents or metabolites could support personalized dosing strategies. Emerging technologies such as microflow LC and high-resolution MS promise even faster analysis and enhanced sensitivity, paving the way for point-of-care therapeutic monitoring.

Conclusion

A rapid, fully automated HILIC-MS/MS method was established for reliable quantification of key aminoglycoside antibiotics and vancomycin. The approach demonstrates strong linearity, accuracy, precision, and high recovery, offering significant advantages over immunoassays and fitting seamlessly into high-throughput clinical laboratory environments.