Fully Automated Simultaneous Analysis of Cephem Antibiotics in Serum by LC-MS/MS

Significance of the Topic

Rapid and reliable quantification of cephem antibiotics in serum is critical for therapeutic drug monitoring, antimicrobial stewardship and quality control in pharmaceutical settings. Automated workflows minimize manual errors and accelerate turnaround times in clinical and industrial laboratories.

Objectives and Study Overview

This application note describes the development of a fully automated sample preparation and LC-MS/MS method for simultaneous analysis of 12 key cephem antibiotics in plasma. The goals were to streamline pretreatment, achieve high throughput and maintain robust analytical performance.

Methodology

Sample pretreatment was performed on the CLAM-2030 module. The procedure involved:

• Addition of 20 µL 75 % isopropanol
• Dispense 20 µL plasma sample and 10 µL internal standard mix
• Injection of 100 µL methanol and mixing for protein precipitation
• Suction filtration through an immunoaffinity trap column

The eluate was transferred directly to the LC-MS/MS system for analysis.

Used Instrumentation

• CLAM-2030 fully automated pretreatment module
• Nexera X2 UHPLC with DOSIMMUNE trap and analytical columns
• LCMS-8060 triple quadrupole mass spectrometer (ESI positive, MRM mode)

Main Results and Discussion

Calibration standards ranging from 0.01 to 5 µg/mL for each of the 12 antibiotics showed excellent linearity (R² ≥ 0.998), accuracy within ±10 % and precision (%RSD) below 15 %. Plasma samples spiked at 0.3 and 3 µg/mL (resulting in measured concentrations of 0.04 and 0.4 µg/mL after dilution) yielded recoveries within 100 ± 15 % and %RSD < 15 %. Total cycle time per sample was 3 minutes for pretreatment and 7 minutes for LC-MS/MS measurement.

Benefits and Practical Applications

• Fully automated workflow reduces manual handling and potential cross-contamination
• High throughput enables up to six replicates per calibrator in minutes
• Applicable to clinical laboratories for drug monitoring and to pharmaceutical QC for residual analysis

Future Trends and Opportunities

Advances may include integration of multiplexed assays to expand antibiotic panels, incorporation of machine learning for data processing, miniaturized modules for point-of-care testing and adaptation to other drug classes for broader clinical and environmental monitoring.

Conclusion

The described automated LC-MS/MS workflow delivers robust, high-throughput quantitation of 12 cephem antibiotics in serum with excellent accuracy, precision and minimal hands-on time, supporting rapid decision-making in clinical and industrial laboratories.

Reference

Yoshikawa K.; Minohata T. Fully Automated Simultaneous Analysis of Cephem Antibiotics in Serum by LC-MS/MS. Shimadzu Application News 01-00444-EN, First Edition, Mar. 2023.