Quantitative Analysis of -Lactam Antibiotics in Human Plasma by High Sensitivity LC/MS/MS Method

Importance of the Topic

The accurate quantitation of β-lactam antibiotics in human plasma is essential for therapeutic drug monitoring, optimizing dosage, minimizing resistance development and improving patient outcomes.

Study Objectives and Overview

This work describes a high-sensitivity LC/MS/MS method for the simultaneous quantitation of five β-lactam antibiotics – meropenem, tazobactam, piperacillin, cefepime and ceftazidime – in human plasma. The goal was to develop a fast, robust assay with minimal sample volume, simple preparation and reliable performance metrics.

Methodology and Instrumentation

A protein precipitation protocol was applied by adding acetonitrile:methanol (1:1) to plasma, followed by vortexing, centrifugation and filtration. A 2 μL aliquot of supernatant was injected onto a Kinetex C18 column under a 5.5-minute gradient. Detection employed a Shimadzu LCMS-8060 triple quadrupole with heated ESI in positive MRM mode. Stable isotope-labeled analogs served as internal standards for each analyte (MER-d6 for tazobactam).

Instrumental Details

• LC: Kinetex 1.7 μm C18, 100×2.1 mm, 0.5 mL/min, 40 °C
• Mobile Phases: 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B)
• Gradient: 5% B (0–0.2 min) → 90% B (3.5–4.0 min) → 5% B (4.1–5.5 min)
• MS: MRM positive mode, block 400 °C, nebulizing gas N2 3.0 L/min, drying gas N2 5.0 L/min

Main Results and Discussion

Calibration curves (20–4000 ng/mL) showed excellent linearity (R2 > 0.997). Accuracy ranged from 87% to 109% across concentrations, with repeatability (%RSD) generally below 6%. Recoveries exceeded 86% for all analytes. Initial matrix effects were notable for ceftazidime (33–40%) and tazobactam (128–149%) but improved (62–85% and 95–109%, respectively) after 2.5-fold dilution.

Benefits and Practical Applications

• Rapid analysis with total run time under 6 minutes
• High sensitivity (LOQs 0.7–6.0 ng/mL) with 2 μL injection volume
• Simple protein crash sample prep supports high throughput
• Isotope dilution ensures specificity and robust quantitation

Future Trends and Opportunities

Emerging advances may include ultra-high-pressure LC for further speed, HRMS for broader screening, microfluidic sample handling to reduce volume, and automation to integrate direct sampling and data processing in clinical settings.

Conclusion

A fast, sensitive LC/MS/MS assay was established for five β-lactam antibiotics in plasma, demonstrating linearity, accuracy, precision, and robustness. The method supports therapeutic drug monitoring with minimal sample and system maintenance burdens.

References

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