Reliable Determination of Sulfonamides in Water Using UHPLC Tandem Mass Spectrometry

Importance of the topic

Antibiotic residues, notably sulfonamides, are prevalent environmental contaminants due to extensive use in human and veterinary medicine. Their persistence leads to selective pressure for resistant bacteria and ecological imbalances. Reliable quantification of trace-level sulfonamides in surface water supports risk assessment, regulatory compliance, and mitigation strategies.

Objectives and overview of the study

This application note aims to develop and validate a robust analytical method for the simultaneous determination of 18 sulfonamides and one co-existent substance (trimethoprim) in surface waters. The approach combines solid-phase extraction (SPE) with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using Agilent 1290 Infinity II systems coupled to either a 6470 or an Ultivo mass spectrometer.

Methodology and instrumentation

• Sample preparation:
  • Adjust water sample to pH 4–7 and add EDTA (0.5 g/L).
  • Spike isotopically labeled surrogates (IS1–IS3) and filter.
  • Load on Agilent Bond Elut PPL SPE cartridge; wash and vacuum-dry.
  • Elute analytes with methanol containing 2% ammonia; evaporate to ≤0.5 mL.
  • Add injection internal standard (IS4) and reconstitute to 1 mL; centrifuge.
• UHPLC conditions:
  • Column: Agilent InfinityLab Poroshell C18 (2.1 × 150 mm, 2.7 µm).
  • Mobile phase A: 0.2% acetic acid in water; B: methanol.
  • Gradient: 10%–95% B over 16 min; flow rate 0.30 mL/min; T = 35 °C; inj. vol. = 2 µL.
• MS/MS conditions:
  • Instruments: Agilent 6470 or Ultivo with Jet Stream source (positive mode).
  • MRM acquisition; capillary voltage 3500 V; gas temperatures 325–350 °C; gas flows N₂.
  • Optimized transitions and collision energies for 19 analytes and internal standards.

Main results and discussion

• Linearity: Calibration range 0.5–100 µg/L with R² > 0.996 for all analytes.
• Sensitivity: LOQs at low ppt levels; S/N > 10 on 6470 MS for all compounds; most S/N > 10 on Ultivo.
• Recoveries in pure water at 20–400 ng/L spiking: 70%–96% (n=6), RSD < 15%.
• Surface water recovery at 20 ng/L: 80%–90%, RSD < 20% (n=4).
• SPE elution study: Complete elution in first two 2 mL fractions; optimum pH 4–7.

Benefits and practical applications

• Sensitive and accurate quantitation of trace sulfonamides in diverse water matrices.
• Meets regulatory standards (e.g., China GB/T 27417-2017, US EPA 1694).
• Applicable to routine environmental monitoring and water quality assessment.

Future trends and possibilities

• Extension to broader classes of antibiotics and transformation products.
• Integration with high-resolution MS for non-target screening.
• Automation of SPE workflows and miniaturization for high-throughput analysis.

Conclusion

The developed SPE-UHPLC-MS/MS method provides robust, reproducible, and high-throughput analysis of 19 sulfonamide antibiotics in water, enabling reliable environmental monitoring and regulatory compliance.

Used instrumentation

• Agilent 1290 Infinity II UHPLC system.
• Agilent 6470 Triple Quadrupole MS and Agilent Ultivo LC/MS/MS.
• Agilent Bond Elut PPL SPE cartridges.

References

1. Danner M-C. et al. Antibiotic Pollution in Surface Fresh Waters: Occurrence and Effects. Sci. Total Environ. 2019, 644, 793–804.
2. Zhang Q-Q. et al. Comprehensive evaluation of antibiotics emission and fate in the river basins of China. Environ. Sci. Technol. 2015, 49, 6772–6782.
3. Wang D. et al. Pharmaceutical and Personal Care Products in the Surface Water of China: A Review. Chin. Sci. Bull. (Chin.) 2014, 59, 743–751.
4. US EPA. Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS; 2007.
5. China GB/T 27417-2017: Conformity Assessment—Guidance on Validation and Verification of Chemical Analysis Methods; 2017.