Highly sensitive quantitative analysis of Amoxicillin and Clavulanic acid from plasma using LC/MS/MS

Importance of the topic

Accurate quantitation of amoxicillin and clavulanic acid at trace levels in plasma is essential for pharmacokinetic profiling, therapeutic monitoring and drug development. Their combination enhances antibacterial efficacy and combating resistance, making sensitive analytical methods crucial for clinical and research laboratories.

Objectives and study overview

This study aimed to develop and validate a fast, highly sensitive LC/MS/MS assay for the simultaneous determination of amoxicillin and clavulanic acid in human plasma. Key objectives included:

• Achieving low limits of quantification
• Minimizing sample preparation steps
• Utilizing ultra-fast polarity switching for dual-mode detection

Methodology and instrumentation

Sample preparation involved adding sodium carbonate to plasma, extracting with diethyl ether/hexane, evaporating the organic layer and reconstituting in water/acetonitrile. Calibration curves ranged from 1 to 100 ng/mL for amoxicillin and 3 to 100 ng/mL for clavulanic acid. The analytical system comprised:

• UHPLC Nexera with Shim-pack XR-ODS column (50×3 mm, 2.2 μm)
• Mobile phase A: 10 mM ammonium formate in water; B: acetonitrile; gradient elution
• Flow rate 0.3 mL/min, column temperature 40 °C, injection volume 30 μL
• Shimadzu LCMS-8040 triple quadrupole with ESI source
• Ultra-fast scanning (15 000 u/s) and polarity switching (15 ms)

Main results and discussion

Retention times were approximately 1.06 min for amoxicillin and 0.99 min for clavulanic acid without full chromatographic separation. Linearity was excellent in both aqueous and matrix-matched standards (r2 > 0.993). Limits of quantification were 1 ng/mL for amoxicillin and 3 ng/mL for clavulanic acid. Accuracy ranged between 93 and 109% and intra-day precision (RSD) remained below 15%. No interfering peaks were observed in blank plasma.

Benefits and practical applications

The method offers rapid throughput with minimal sample preparation and robust performance in complex biological matrices. It is suited for high-throughput pharmacokinetic studies, bioequivalence testing and routine therapeutic drug monitoring.

Future trends and opportunities

Continued improvements in mass spectrometer scan speeds and ionization sources may allow even lower detection limits. Integration with automated sample handling and microflow LC could increase throughput. Adaptation to other β-lactam/β-lactamase inhibitor combinations is a promising extension.

Conclusion

The validated LC/MS/MS assay using Shimadzu Nexera and LCMS-8040 achieves sensitive, precise and rapid determination of amoxicillin and clavulanic acid in plasma, fulfilling stringent demands for pharmacokinetic and clinical studies.

Reference

• Avinash Gaikwad et al., Journal of Pharmacy Research 6(8):804-812 (2013)
• Rajinder Singh Gujral et al., International Journal of Biomedical Science 6(4):335-343 (2010)