On-line Enrichment HTLC/MS/MS Assay for Multiple Classes of Antibiotics in Environmental Water Sources

Significance of the Topic

Environmental contamination by trace antibiotics threatens public health and ecosystems. Monitoring these compounds at ultra-trace levels demands efficient sample preparation and sensitive detection. On-line turbulent-flow high-throughput liquid chromatography coupled with tandem mass spectrometry (HTLC-MS/MS) streamlines analysis of multiple antibiotic classes in environmental waters with minimal prep time and high selectivity.

Objectives and Study Overview

The study aims to develop and validate an on-line preconcentration and extraction method for screening various antibiotics in surface water using HTLC-MS/MS with highly selective reaction monitoring.

Methodology and Instrumentation

Water samples (1 mL) were pretreated with Na2EDTA to prevent antibiotic binding, then directly injected onto a TurboFlow Cyclone MAX column for on-line enrichment at 2.0 mL/min. Analytes were eluted with 50% acetonitrile/0.1% formic acid and refocused on a 4.6×100 mm Hypersil GOLD analytical column under a gradient of ammonium bicarbonate, acetonitrile, formic acid, and TFA at 1.2 mL/min. Detection employed a TSQ Quantum Ultra mass spectrometer in positive electrospray ionization, using both unit and high-resolution SRM (0.7 and 0.15 Da FWHM) for two transitions per compound. Key instrumentation:

• On-line extraction: Aria TLX-2 with TurboFlow Cyclone MAX (0.5×50 mm)
• Analytical LC: Thermo Hypersil GOLD, 4.6×100 mm, 3 μm
• Mass spectrometer: TSQ Quantum Ultra, positive ESI, ion transfer tube at 375°C

Main Results and Discussion

Thirteen antibiotics spanning sulfonamides, tetracyclines, and macrolides achieved limits of quantitation between 0.5 and 5 pg/mL with 1 mL injections. Calibration exhibited R2 ≥ 0.990 for most compounds (linear 1/x weighting; sulfamethoxazole and sulfamethizole required quadratic fits). Spike recovery in surface water at 25 pg/mL revealed minor matrix suppression for sulfonamides, slight enhancement for macrolides, and significant suppression for tetracyclines—likely due to metal binding. Environmental screening detected sulfamethoxazole, erythromycin, tylosin, and dehydroerythromycin at ~2 pg/mL in Lake Ontario samples, confirmed by dual SRM transitions.

Benefits and Practical Applications

• High throughput: 1 mL on-line injection reduces sample prep and analysis time from hours to minutes.
• Enhanced selectivity: dual H-SRM transitions deliver confident identification at ultra-trace levels.
• Broad applicability: simultaneous multi-class antibiotic screening in environmental waters.

Future Trends and Opportunities

Advancements may include coupling on-line enrichment with high-resolution mass spectrometry for wider contaminant coverage, automated data processing pipelines for large-scale monitoring, and miniaturized or field-deployable systems enabling real-time water quality assessment.

Conclusion

The presented HTLC-MS/MS method offers a fast, sensitive, and selective approach for monitoring trace antibiotics in environmental water sources, significantly improving laboratory throughput and data confidence.

References

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