LC-MS/MS Analysis of Anti-Infectives In Raw and Treated Sewage

Importance of the Topic

Anti-infective agents—including synthetic antimicrobials and natural or semi-synthetic antibiotics—are routinely used in human and veterinary medicine. A significant fraction of these compounds is excreted unchanged and enters municipal wastewater. Conventional treatment processes often fail to fully remove these substances, leading to their release into surface waters. Monitoring trace concentrations of anti-infectives in sewage is essential to assess environmental exposure risks and potential ecological effects, including microbial resistance development.

Objectives and Overview of the Study

This work focused on developing a sensitive and reliable analytical workflow to detect and quantify six commonly prescribed anti-infectives in both raw influent and treated effluent from a large municipal wastewater plant in Montréal, Canada. Key goals included:

• Achieving quantification at the lower nanogram-per-liter level.
• Employing two specific single reaction monitoring (SRM) transitions per analyte and verifying peak-area ratios to minimize false positives.

Methodology and Instrumentation

Samples of raw sewage (north and south influents) and 24-hour composite effluent were collected at the Montréal wastewater treatment plant, which applies only physico-chemical processes. Each 250 mL sample was filtered (1.2 µm and 0.45 µm), acidified to pH 3, and spiked with 500 ng L⁻¹ of a surrogate standard (pyrimethamine). A tandem solid-phase extraction (SPE) using reversed-phase and mixed-mode polymeric cartridges concentrated the analytes. Eluates were evaporated under nitrogen and reconstituted in 0.1% formic acid in a water/MeOH/ACN mixture containing internal standards (diaveridine, lomefloxacin, josamycin). Chromatographic separation was performed on a BetaBasic C18 column (50 × 2.1 mm, 3 µm) using a Thermo Scientific Surveyor HPLC with a gradient of 0.1% formic acid in water and MeOH/ACN (1:1). Detection employed a TSQ Quantum Ultra triple-quadrupole mass spectrometer in positive electrospray ionization mode, with two SRM transitions and defined collision energies and tube-lens voltages for each compound.

Main Results and Discussion

The method exhibited linear responses (r² ≥ 0.99) across 25–1000 ng L⁻¹ despite complex wastewater matrices. Limits of detection spanned 0.3–22 ng L⁻¹. Tandem SPE recoveries ranged from 60% to 100% for all six anti-infectives. Dual SRM transitions and peak-ratio confirmation provided high selectivity, preventing false positives (RSD <10%, peak-ratio deviations <20% for most analytes). Measured concentrations in raw and treated samples varied between 39 ± 1 and 276 ± 7 ng L⁻¹. Estimated daily mass flows discharged into the St. Lawrence River ranged from 118 to 830 g per compound, underlining significant environmental loading despite low ambient concentrations.

Contributions and Practical Applications

This analytical approach enables robust monitoring of anti-infectives at trace levels in complex wastewater matrices. It supports environmental surveillance programs, regulatory compliance assessments, and the evaluation of treatment plant performance regarding pharmaceutical removal. The method’s sensitivity and specificity make it applicable to QA/QC in environmental laboratories and research settings.

Future Trends and Potential Uses

• Extension to a broader range of pharmaceutical classes and emerging contaminants.
• Integration with high-resolution mass spectrometry for non-target screening.
• Application in longitudinal studies to assess seasonal and operational impacts on removal efficiencies.
• Coupling environmental monitoring data with ecotoxicological assessments to evaluate ecosystem risks.

Conclusion

The developed LC-MS/MS method, combining tandem SPE and dual-transition SRM on a triple-quadrupole instrument, effectively quantifies six anti-infectives in raw and treated sewage at nanogram-per-liter levels. The approach offers high selectivity, low detection limits, and reliable quantification, making it a valuable tool for environmental monitoring of pharmaceutical residues.

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