Analysis of Amoxicillin using the LCMS-2010EV and the LCMS-IT-TOF

Meaning of the Topic

Amoxicillin is a widely used β-lactam antibiotic effective against various bacterial pathogens. Accurate analysis of this compound is crucial for quality control in pharmaceutical production, monitoring therapeutic levels in clinical settings, and studying degradation pathways. Mass spectrometric methods combined with liquid chromatography provide both separation and structural information, supporting regulatory compliance and research into new formulation strategies.

Objectives and Study Overview

This work compares the performance of two mass spectrometers—LCMS-2010EV and LCMS-IT-TOF—for the analysis of amoxicillin. The study aims to (1) establish chromatographic conditions for reproducible separation, (2) assess fragmentation patterns and sensitivity on both instruments, and (3) evaluate the mass accuracy and structural elucidation capabilities of the trap/time-of-flight system.

Methodology and Instrumentation

Sample solutions of amoxicillin were prepared in methanol–acetonitrile mixtures at 1–1.25 mg/mL. A Shim-pack VP-ODS column (4.6 × 150 mm) was used with a water/acetonitrile gradient containing 0.1% formic acid. Key LC parameters included a 0–30 min linear gradient from aqueous to organic-rich mobile phase and a 0.3 mL/min flow rate.

• Injection volume: 1 µL (LCMS-2010EV), 0.5 µL (LCMS-IT-TOF)
• ESI probe voltage: +4.5 kV
• CDL/block temperatures: 200–250 °C
• Nebulizing gas: 1.5 L/min

Used Instrumentation

• Shimadzu LCMS-2010EV with ESI source
• Shimadzu LCMS-IT-TOF enabling MSn and high-accuracy measurements

Main Results and Discussion

Both instruments produced a clear protonated amoxicillin ion at m/z 366.11. On the LCMS-2010EV, increasing Q-array voltage induced in-source dissociation, revealing characteristic fragment ions at m/z 349, 279, 261, and 205. The LCMS-IT-TOF delivered similar fragmentation but with enhanced mass accuracy (≤3.4 ppm) for precursor and product ions, facilitating unambiguous assignment of elemental compositions.

Multistage MS experiments on the IT-TOF (MS2 and MS3) confirmed the structure of key fragments, supporting detailed pathway elucidation of amoxicillin breakdown.

Benefits and Practical Applications

These methodologies allow pharmaceutical laboratories to:

• Perform robust quality control of amoxicillin formulations
• Study degradation products to ensure drug safety
• Leverage high-accuracy MSn data for impurity profiling and structural verification

Future Trends and Potential Applications

Advances may include coupling high-resolution mass spectrometers with rapid UPLC methods for faster throughput, integration with automation platforms for large-scale screening, and application of data-independent acquisition to capture broader fragmentation information. Emerging hybrid instruments could further improve confidence in structure elucidation without external standards.

Conclusion

This comparative study demonstrates that both LCMS-2010EV and LCMS-IT-TOF deliver reliable amoxicillin analysis, with the IT-TOF offering superior mass accuracy and multistage fragmentation. These capabilities enhance quality assurance and structural investigations in pharmaceutical research.

Reference

1. Näegele E., Moritz R. Structure elucidation of degradation products of the antibiotic amoxicillin with ion trap MSn and accurate mass determination by ESI TOF. J. Am. Soc. Mass Spectrom. 2005;16:1670–1676.