Analysis of Macrolides and Three Other Antibiotic Classes at Low ppt Levels in WWTP Effluent and Surface Waters by LC-MS/MS

Importance of the Topic

Antibiotic residues released from wastewater treatment plants into rivers and lakes pose a growing ecotoxicological concern and contribute to the emergence of antimicrobial resistance in aquatic microbial communities. Regulatory initiatives such as the EU watch list (EU 2015/495) highlight the need for reliable trace-level monitoring of key antibiotics, including macrolides, in environmental waters.

Goals and Study Overview

This study aimed to develop and fully validate a sensitive LC-MS/MS method to detect and quantify three macrolides (azithromycin, clarithromycin, erythromycin) alongside six additional antibiotics at low parts-per-trillion levels in wastewater treatment plant (WWTP) effluent and downstream surface waters. The approach builds on a U.S. EPA-published protocol, extending its scope and improving performance.

Methodology

• Sample Preparation: 50 mL water samples spiked with target analytes were processed by off-line solid-phase extraction (SPE) using 200 mg Oasis HLB cartridges.
• SPE Workflow: Cartridges were conditioned with methanol and water, loaded under controlled pressure, air-dried for reproducible evaporation, eluted with methanol, and evaporated to dryness.
• Reconstitution and Injection: Dried extracts were reconstituted in 1 mL of methanol/water and 1 µL was injected.
• Chromatography: Two parallel ACQUITY UPLC BEH C18 columns (2.1 mm×50 mm, 1.7 µm) were operated under complementary acidic and basic gradients to optimize selectivity and sensitivity.
• Mass Spectrometry: A Xevo TQ-S triple quadrupole mass spectrometer with positive electrospray ionization monitored two MRM transitions per compound.

Instrumentation

• Waters ACQUITY UPLC H-Class System with column manager for rapid switching.
• Waters Xevo TQ-S MS detector.
• Oasis HLB SPE cartridges (200 mg).
• Certified TruView LC-MS vials for trace analysis.

Main Results and Discussion

The method achieved method quantification limits (MQLs) of 0.5–32 ng/L for the nine antibiotics with linearity coefficients (R²) ≥0.997. Absolute SPE recoveries ranged from 48% to 91%, and overall method recoveries reached up to 110% in humic-rich waters. Analysis of WWTP effluent and downstream lake samples detected all three macrolides at levels between 2.6 and 215 ng/L. Penicillin G/V and fluoroquinolones were not observed, likely due to rapid β-lactam degradation and sorption of quinolones to sludge. The dual-pH column strategy enhanced chromatographic separation and sensitivity without compromising throughput.

Benefits and Practical Applications

• Trace-level detection well below regulatory limits allows comprehensive environmental surveillance.
• Validated SPE-UPLC-MS/MS workflow supports routine monitoring and research studies.
• Parallel column pH operation offers flexibility to add new analytes with minimal re-optimization.
• High extraction efficiency and reproducible drying procedures ensure consistent performance.

Future Trends and Opportunities

• Incorporation of high-resolution MS for non-target and suspect screening of emerging contaminants.
• Automation of SPE and online coupling to increase throughput and reduce manual handling.
• Miniaturized and field-deployable sampling platforms for in situ monitoring.
• Multiclass methods expanded to cover antiviral, antifungal, and other pharmaceutical residues.

Conclusion

The presented LC-MS/MS method delivers robust, sensitive, and flexible analysis of multiple antibiotic classes in environmental waters at low ppt levels. Full validation in line with EPA guidelines and successful application to WWTP effluent and surface water demonstrate its suitability for routine monitoring and environmental risk assessment.

References

• Commission Implementing Decision (EU) 2015/495 of 20 March 2015. Official Journal of the European Union.
• Svahn O. Applied Environmental Analytical Chemistry for Monitoring and Measures Against Antibiotics and Drug Residues in Vattenriket. Lund University Thesis, 2016.
• Svahn O., Björklund E. Increased electrospray ionization intensities and expanded chromatographic possibilities for emerging contaminants using mobile phases of different pH. Journal of Chromatography B, 1033:1–10, 2016.
• EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC-MS/MS, 2007.