Ultrafast Analysis of Lamotrigine, Zonisamide, Gabapentin, Pregabalin, MHD, and Levetiracetam in Serum by the Agilent RapidFire High-Throughput Mass Spectrometry System

Significance of the topic

The analysis of antiepileptic drugs in serum is essential for therapeutic drug monitoring, pharmacokinetic studies, and clinical research. Traditional LC/MS/MS and HPLC methods offer high selectivity but are limited by long run times, high solvent use, and low throughput. An ultrafast SPE/MS/MS approach addresses these challenges by reducing analysis time and resource consumption, enabling rapid decision-making in clinical and research settings.

Objectives and Study Overview

This study aimed to develop and validate a simultaneous quantitation method for six drugs (lamotrigine, zonisamide, gabapentin, pregabalin, MHD, and levetiracetam) in human serum using the Agilent RapidFire high-throughput mass spectrometry system. Key goals included achieving a broad linear calibration range (1–100 µg/mL), high accuracy and precision, negligible carryover, and a sample throughput exceeding 250 samples per hour.

Methodology and Used Instrumentation

Sample preparation involved protein precipitation with methanol and acetonitrile, followed by a 1:50 dilution in water containing isotopic internal standards. The diluted samples were loaded onto a reversed-phase C18 RapidFire cartridge and eluted directly into an Agilent 6460 triple quadrupole mass spectrometer using rapid SPE/MS/MS cycles. MassHunter software controlled data acquisition and performed quantitation via multiple reaction monitoring (MRM) with 1/X weighted linear regression.

Used Instrumentation

• Agilent RapidFire 360 high-throughput SPE system
• Agilent 6460 Triple Quadrupole Mass Spectrometer
• Agilent MassHunter Triple Quadrupole Acquisition, Qualitative Analysis, Quantitative Analysis, and RapidFire Acquisition Software

Main Results and Discussion

The method demonstrated a 14-second cycle time per injection, resulting in over 250 analyses per hour. Calibration curves for all six analytes exhibited high linearity (R2 > 0.995). Intra- and interday accuracy remained within ±7 %, and precision (CV) was below 7 % across QC levels. Carryover was negligible (0 %), and reproducibility over more than 2,000 sequential injections showed CVs of 1.6–7 %, confirming system robustness.

Benefits and Practical Applications

This ultrafast SPE/MS/MS approach offers more than tenfold savings in analysis time and solvent usage compared to conventional LC/MS/MS. It supports high-throughput therapeutic drug monitoring, large-scale pharmacokinetic profiling, clinical trials, and quality control in pharmaceutical development.

Future Trends and Potential Applications

Advancements may include expanded drug panels, integration with automated sample handling, miniaturized SPE cartridges, and real-time data processing aided by artificial intelligence. Such developments will further enhance throughput, reduce costs, and enable broader adoption in clinical laboratories.

Conclusion

The Agilent RapidFire SPE/MS/MS method provides a rapid, accurate, and high-throughput solution for quantifying six antiepileptic drugs in human serum. With a 14-second analysis time and robust performance, this approach outperforms traditional LC/MS/MS in speed and efficiency, making it well suited for demanding clinical and research environments.

References

• Youssef M., Miller V.P. Ultrafast Analysis of Lamotrigine, Zonisamide, Gabapentin, Pregabalin, MHD, and Levetiracetam in Serum by the Agilent RapidFire High-Throughput Mass Spectrometry System. Agilent Technologies Application Note, July 2014.