Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Significance of the Topic

Therapeutic drug monitoring of antiepileptic drugs is crucial for optimizing dosing, improving patient outcomes, and minimizing adverse effects. Rapid and accurate quantification of multiple AEDs in plasma supports personalized treatment plans in clinical practice.

Objectives and Study Overview

This study sought to develop a high-speed, sensitive UHPLC–MS/MS assay for simultaneous measurement of seventeen antiepileptic drugs in plasma. An automated UHPLC method scouting system was employed to streamline chromatographic method development and reduce optimization time.

Experimental Methodology and Instrumentation

The analytical platform combined a Nexera X2 Method Scouting System with an LCMS-8050 triple quadrupole mass spectrometer (Shimadzu). Automated scouting evaluated 36 combinations of mobile phases (including aqueous buffers with ammonium acetate, formate or formic acid) and four column chemistries (ODS-4, XB-C18, PFP, HS F5). Electrospray ionization in positive and negative modes was used. MRM transitions and compound-dependent voltages were optimized via flow injection analysis. Sample preparation involved protein precipitation with acetonitrile followed by direct dilution (100–10 000×) and 1 µL injection.

Main Results and Discussion

The optimal separation used an Inertsil ODS-4 column (2.1×50 mm, 2 µm) with a 10 mM ammonium acetate–methanol gradient over 10 minutes at 0.4 mL/min. All 17 compounds, including carbamazepine, lamotrigine, levetiracetam, phenobarbital and phenytoin, were baseline separated within a 10 minute cycle. Calibration curves showed linearity with correlation coefficients above 0.995 across clinical concentration ranges. Quality control samples at low, medium and high levels exhibited precision and accuracy within ±15 %.

Benefits and Practical Applications

• Automated method scouting accelerates chromatographic optimization without extensive manual testing
• Simple protein precipitation and dilution workflow reduces sample preparation time
• Short 10 minute run time and minimal injection volume (1 µL) support high throughput and reduced solvent use
• Reliable quantification of 17 AEDs enhances comprehensive therapeutic drug monitoring in clinical laboratories

Future Trends and Opportunities

Advances in AI-driven method optimization and miniaturized UHPLC systems may further decrease development time and solvent consumption. Integration of online sample preparation and direct-to-MS interfaces could enable near real-time point-of-care monitoring. Expansion of automated scouting to other drug classes and biomarkers will broaden clinical applications.

Conclusion

The automated UHPLC–MS/MS approach enabled rapid development and robust quantification of multiple antiepileptic drugs with excellent linearity, precision and accuracy. This workflow improves efficiency and throughput for therapeutic drug monitoring in clinical settings.

References

Miho Kawashima et al., Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS, ASMS 2014 ThP672