Analysis of Four β-Lactam Antibiotics in Water Using an Agilent 1290 Infinity II LC and an Agilent 6470 Triple Quadrupole LC/MS with Direct Injection and Online SPE

Importance of the Topic

Environmental monitoring of antibiotics is critical due to widespread use of β-lactam drugs in human and veterinary medicine, which can foster resistance and affect water quality. Sensitive detection at ng/L levels is essential to understand their fate in surface water and wastewater treatment effluents.

Study Objectives and Overview

This work aimed to establish and compare two workflows—direct aqueous injection and automated online SPE—on an Agilent 1290 Infinity II UHPLC system coupled to a 6470 Triple Quadrupole LC/MS. The target analytes were amoxicillin, ampicillin, piperacillin, and penicillin V at trace concentrations in water.

Methodology and Instrumentation

The separation employed a 12-min reversed-phase gradient on a ZORBAX EclipsePlus C18 column (50×2.1 mm, 1.8 μm) using mobile phase A (water with ammonia, pH 8) and B (acetonitrile with formic acid). Two sample introduction modes were evaluated:

• Direct injection: 100 µL sample injected with minimized delay volume for rapid analysis.
• Online SPE: enrichment of 1.5 mL sample on two PLRP-S cartridges, followed by backflush elution into the UHPLC gradient.

Used Instrumentation:

• Agilent 1290 Infinity II Multisampler and Flexible Cube with integrated valve drives.
• Agilent 1260 Infinity II Isocratic Pump for SPE loading.
• Agilent 6470 Triple Quadrupole LC/MS with Jet Stream ESI and dynamic MRM.

Main Results and Discussion

Direct injection achieved baseline separation in 12 min, with LODs of 1–5 ng/L, RSDs below 2%, and linear response up to 20 µg/L. Online SPE improved sensitivity to LODs of 0.2–0.5 ng/L for ampicillin, piperacillin, and penicillin V, with recoveries from 52% to 94% and RSDs of 1.9–5.3%. Amoxicillin exhibited only 6.5% recovery on SPE and was excluded from the enriched workflow. Retention times remained consistent across modes due to optimized low-volume connections.

Benefits and Practical Applications of the Method

This hybrid approach allows seamless switching between direct injection and SPE without hardware reconfiguration, combining high throughput with ultratrace sensitivity. It is well suited for routine analysis of β-lactam residues in environmental monitoring and water quality assessment.

Future Trends and Opportunities

Further work may focus on tailored SPE phases for highly polar antibiotics, expansion to broader pharmaceutical panels, integration with automated field sampling, and application to long-term ecological surveys and antibiotic resistance tracking.

Conclusion

The described UHPLC/MS method delivers robust, sensitive, and flexible quantitation of key β-lactam antibiotics in water. It provides low detection limits, precise quantitation, and rapid mode switching, addressing the needs of environmental and ecotoxicological research.

References

• S. Shaikh et al., “Antibiotic resistance and extended spectrum beta-lactamases: Types, epidemiology and treatment,” Saudi Journal of Biological Sciences 22:90–101 (2015).
• A. Küster et al., “Antibiotics in the environment – effects and side-effects,” German Federal Environment Agency UMID 01:18–28 (2013).
• J. M. Cha, S. Yang, K. H. Carlson, “Trace determination of β-lactam antibiotics in surface water and urban wastewater using LC–ESI–MS/MS,” Journal of Chromatography A 1115:46–57 (2006).