Detection of Trace Level Pharmaceuticals in Drinking Water by Online SPE Enrichment with the Agilent 1200 Infinity Series Online-SPE Solution

Importance of the topic

Rapid and sensitive monitoring of trace-level antibiotics in drinking water is critical for public health and environmental protection. Antibiotic residues can contribute to antibiotic resistance and pose risks even at parts-per-trillion levels, requiring robust analytical techniques to ensure water safety and compliance with regulatory standards.

Objectives and study overview

• Develop an online solid-phase extraction (SPE) coupled with HPLC–MS/MS method to detect 13 major antibiotics at trace levels down to 1 ppt in drinking water.
• Evaluate linearity, precision, recovery, and carryover using Agilent 1200 Infinity Series Online-SPE with a 1290 Flexible Cube and a triple quadrupole mass spectrometer.
• Assess method performance in real water samples from river, tap, and spring sources.

Instrumentation

• Agilent 1200 Infinity Series Online-SPE system with 1290 Infinity Flexible Cube (2-position/10-port valve).
• Agilent 1260 Infinity quaternary pump, 1290 Infinity thermostatted column compartment, and 1260 Infinity autosampler (multidraw).
• Agilent ZORBAX Eclipse Plus C18 analytical column (2.1×150 mm, 3.5 µm).
• Agilent 6460 triple quadrupole LC/MS with Jet Stream technology.
• MassHunter software for data acquisition, qualitative and quantitative analysis.

Methodology

• Sample loading: Automated multidraw injection (1.8 mL) onto two alternating SPE cartridges (PLRP-S) under high backpressure.
• SPE clean-up: Parallel trapping and reconditioning cycles using multiple wash solvents via the Flexible Cube piston pump.
• Chromatography: Gradient elution from 5% to 98% organic (ACN/ammonium formate) at 0.4 mL/min, column at 40 °C, total cycle ~35 min including re-equilibration.
• MS/MS detection: Dynamic multiple reaction monitoring (dMRM) optimized for each compound, employing Jet Stream thermal gradient focusing; LODs of 0.5–5 ppt achieved with signal-to-noise criteria.
• Calibration and validation: Seven-point calibration (100 to 1 ppt), retention time and peak area precision (RSD <6%), recovery 41–109% across antibiotic classes.

Main results and discussion

The method demonstrated excellent linearity (R2 ≥0.995) across a broad polarity range of antibiotics. Limits of quantification reached as low as 1 ppt for most analytes. Retention time RSDs were <0.15% and peak area RSDs <6% for a 50 ppt standard. SPE recoveries ranged from 41% (tetracyclines) to 109% (fluoroquinolones), with most compounds above 70%. Carryover in blank injections after 100 ppt was <0.2%. Spiked matrices (river, tap, spring water at 20 ppt) showed accuracy of 59–104% and area precision RSDs <2%.

Benefits and practical applications

• Fully automated online SPE reduces manual sample preparation and risk of contamination.
• High sensitivity and low LODs enable compliance with stringent regulatory limits for antibiotics in drinking water.
• Modular design allows parallel SPE cartridge operation for increased throughput.
• Robust performance across diverse antibiotic classes and water matrices supports routine environmental monitoring and quality control.

Future trends and applications

• Expansion to additional emerging contaminants such as hormones, pesticides, and personal care products.
• Integration with high-resolution mass spectrometry for non-target screening and compound identification.
• Development of miniaturized or field-deployable online SPE–MS platforms for on-site water quality assessment.
• Implementation of advanced data analytics and machine learning for automated compound detection and quantification.

Conclusion

The Agilent online SPE–HPLC–MS/MS workflow provides a rapid, sensitive, and reproducible solution for trace-level antibiotic analysis in drinking water. The method meets stringent detection requirements and demonstrates robust performance in real-world samples, making it a valuable tool for environmental monitoring and public health protection.

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