High Resolution LC-MS for Screening and Quantitative Analysis of Antibiotics in Drinking Water Using an Orbitrap and Online Sample Preparation

Importance of the Topic

Antibiotic contamination in drinking water poses significant public health and environmental risks. Conventional methods relying on offline sample preparation and triple quadrupole mass spectrometry require large volumes, extended processing times, and can suffer from reproducibility issues. High-resolution accurate mass (HR/AM) LC-MS with online sample preconcentration offers a streamlined workflow, lower detection limits at ppt/sub-ppt levels, and improved confidence in compound identification.

Objectives and Article Overview

This study demonstrates a workflow integrating online preconcentration with high-resolution LC-MS for screening, quantitation, and confirmation of multiple antibiotics in drinking water. Key goals include minimizing sample volume, achieving ppt-level sensitivity, and validating spectral confirmation within a single analytical run.

Methodology

Water samples (1 mL) were directly injected onto a trapping column and backflushed onto an analytical column using a dual-pump Thermo Scientific EQuan MAX system. Reversed-phase chromatography employed water and methanol (0.1% formic acid, 4 mM ammonium formate) with a 15-min gradient. Detection used a Thermo Scientific Exactive Plus Orbitrap mass spectrometer in alternating full-scan (70,000 resolution) and all-ion fragmentation (AIF, 35,000 resolution) modes, with stepped HCD at 30 eV. Calibration curves (1 ppt–10 ppb range) were constructed in TraceFinder with linear or quadratic fits and 1/x weighting.

Instrumentation

• Thermo Scientific EQuan MAX online sample preparation system
• Accela 600 pump for trapping (Hypersil GOLD aQ, 20×2.1 mm, 12 μm)
• Accela 1250 pump for analytical flow (Accucore aQ, 100×2.1 mm, 2.6 μm)
• Exactive Plus Orbitrap mass spectrometer (Thermo Scientific)
• TraceFinder™ 2.1 and ExactFinder™ 2.0 software for quantitation and spectral confirmation

Main Results and Discussion

Limits of quantitation (LOQs) ranged from 0.2 to 40 pg/mL with %RSD ≤15%. Sub-ppt sensitivity was achieved for most antibiotics, as summarized in LOQ data tables. Spectral confirmation using built-in environmental libraries matched diagnostic fragments (e.g., trimethoprim at m/z 291.1446) even at 80 pg/mL, demonstrating robust identification and minimal matrix interference.

Benefits and Practical Applications of the Method

• Eliminates labor-intensive offline sample preparation
• Reduces required sample volume to 1 mL
• Provides high sensitivity and specificity via HR/AM detection
• Enables concurrent quantitation and confirmation within a single run
• Enhances data re-interrogation capability for retrospective analysis

Future Trends and Potential Applications

Advances may include integration with automated field sampling, expansion to multi-residue screening of emerging contaminants, deployment of portable HR-MS platforms, and application of AI-driven data processing for real-time monitoring. Such developments will support broader environmental surveillance, regulatory compliance, and water safety assurance.

Conclusion

The presented online preconcentration HR-AM LC-MS approach offers a rapid, sensitive, and reproducible solution for screening and quantifying antibiotics in drinking water. Combining quantitation and spectral confirmation in one workflow enhances analytical throughput and confidence, addressing key challenges in environmental monitoring.