Measurement of Antibiotics in Environmental Waters using LC-QQQ and fully automated Online Enrichment

Importance of the topic

Antibiotics are extensively used in human and veterinary medicine and ultimately enter wastewater and surface waters. Their persistence at trace levels can contribute to the development of antibiotic resistance and pose risks to ecosystems and human health. Reliable monitoring of these contaminants is critical for environmental surveillance and risk management.

Objectives and Overview

This work describes a method for the simultaneous quantification of 23 antibiotics from nine different drug classes in environmental water samples. The method integrates fully automated online solid phase enrichment with liquid chromatography triple quadrupole mass spectrometry. After validation, the approach was applied to hospital and domestic wastewater, wastewater treatment plant influent and effluent, and river water to demonstrate performance under real-world conditions.

Methodology

• Sample preparation via online SPE using C18 cartridges with in-line automated enrichment.
• Sample pH adjusted to pH 4 for fluoroquinolones and pH 7 for other compounds.
• Loading of 2×900 µL sample at 1 mL/min followed by backflush elution into the LC system.
• Step gradient elution on a C18 analytical column (3.5 µm, 2.1×150 mm) at 0.5 mL/min.
• Mobile phase consisting of water and acetonitrile with 0.1 formic acid.
• Use of deuterated internal standards norfloxacin-d4, amoxicillin-d4, and sulfamethoxazole-d4 for quantitation.

Instrumentation Used

• Agilent 1200 Series liquid chromatograph
• Agilent 6400 Triple Quadrupole Mass Spectrometer with electrospray ionization
• Online SPE enrichment autosampler and high-pressure gradient pumps
• Quaternary loading pump and multiport selection valves for automated flow control

Main Results and Discussion

Linearity was demonstrated from 0.005 to 50 ng on column for most analytes, with slight variations for amoxicillin and selected compounds. Instrument detection limits ranged from 0.5 to 16 pg on column and method detection limits from 1.4 to 15 ng/L in water. Accuracy and precision were within ±20 of nominal values and below 8.5 RSD. Recoveries exceeded 65 for most antibiotics with precision below 15 RSD. Application to real samples revealed varying concentration profiles, with substances such as erythromycin and ciprofloxacin detected at μg/L levels in hospital wastewater and treatment plant effluent.

Benefits and Practical Applications

• Fully automated workflow reduces manual handling and increases throughput.
• High sensitivity enables trace level detection suitable for regulatory monitoring.
• Robust performance across diverse drug classes supports comprehensive surveillance.
• Fast analysis time of approximately 15 minutes per sample enables routine application in QA/QC and research laboratories.

Future Trends and Potential Applications

Advancements may include integration with high-resolution mass spectrometry for identification of transformation products, miniaturized sample preparation to reduce solvent consumption, and implementation of data analytics for trend monitoring. Expansion to other emerging contaminants and coupling with online oxidation or biodegradation modules could further enhance environmental assessments.

Conclusion

The described LC-QQQ method with automated online SPE offers a reliable, sensitive, and efficient approach for monitoring a broad range of antibiotics in environmental waters. Its strong analytical performance and adaptability to routine workflows make it a valuable tool for environmental monitoring and public health protection.

References

Agilent Technologies Inc Publication Number 5990-6373EN, September 2010.