Quantification of 27 antiepileptics in human plasma by LC-HRAM-MS for clinical research

Significance of the topic

Antiepileptic drugs (AEDs) require precise monitoring in clinical research to optimize dosing, assess therapeutic compliance, and minimize toxicity. Quantifying multiple AEDs at varied plasma concentrations demands robust selectivity, sensitivity, and throughput. High-resolution LC-MS methods address these needs by offering accurate identification and quantitation of diverse compounds in complex matrices.

Goals and study overview

This work describes the development and validation of a liquid chromatography–high-resolution accurate mass spectrometry (LC-HRAM-MS) method to quantify 27 antiepileptic drugs and metabolites in human plasma. Objectives included achieving low limits of quantification (LLOQs), broad linear dynamic range, minimal carryover, and acceptable accuracy and precision for clinical research applications.

Methodology and Instrumentation

• Sample preparation involved protein precipitation of 50 µL plasma with 100 µL acetonitrile containing isotopically labeled internal standards, followed by vortexing, centrifugation, and 10-fold dilution in aqueous mobile phase.
• Chromatography was performed on a Vanquish Flex UHPLC system with a Hypersil GOLD 2.1 × 50 mm, 1.9 µm column at 40 °C, flow 0.4 mL/min, and a 5.5 min gradient from 2 to 98% methanol (0.1% formic acid).
• Detection used a Thermo Scientific Orbitrap Exploris 120 mass spectrometer with Heated ESI in positive and negative modes. Full MS-ddMS2 acquisition at 60 000 resolution (m/z 40–400) ensured selectivity.

Main results and discussion

• All 27 analytes showed linear calibration with 1/x weighting down to LLOQs. LLOQs ranged from 0.011 ng/mL (tiagabine) up to 8.56 ng/mL (valproic acid).
• Back-calculated concentrations exhibited bias within ±15% across calibration points. Maximum carryover observed was 0.2% for gabapentin and pregabalin; other compounds showed negligible carryover.
• Accuracy testing using two QC levels yielded biases between –8.8% and +11.0%. Intra-assay precision (%CV) remained below 9.1% for all analytes; inter-assay precision was under 7.7%.

Benefits and practical applications

• Rapid sample preparation supports high throughput in clinical labs.
• High resolution MS enhances selectivity, reducing interferences and improving confidence in low-level quantitation.
• Wide calibration ranges and low LLOQs enable simultaneous monitoring of multiple AEDs, facilitating pharmacokinetic and therapeutic drug monitoring studies.

Future trends and applications

Advances in LC-HRAM-MS technology will further improve multiplexed drug panels and shorten analysis times. Integration with automated sample preparation and cloud-based data analytics could streamline workflow and support personalized dosing strategies. Expanding to cover novel AEDs and their metabolites will enhance clinical pharmacology research and therapeutic monitoring.

Conclusion

A robust UHPLC–Orbitrap Exploris 120 method was established for simultaneous quantification of 27 antiepileptics in human plasma. The protocol demonstrated excellent linearity, sensitivity, accuracy, and precision, meeting clinical research requirements for therapeutic drug monitoring.

References

• Barcenas M. Quantification of 27 antiepileptics in human plasma by LC-HRAM-MS for clinical research. Thermo Fisher Scientific Technical Note 000110, 2021.