Quantification of antiepileptics in human plasma or serum by LC-HRAM(MS) for clinical research

Significance of the Topic

Antiepileptic drugs (AEDs) are essential for the management of epilepsy, a chronic neurological disorder marked by recurrent seizures. Accurate measurement of AED levels in plasma or serum is critical for guiding therapy, assessing patient adherence, minimizing toxicity, and supporting clinical research. High-resolution accurate-mass (HRAM) mass spectrometry offers both sensitive quantification and the potential for detailed qualitative analyses, increasing confidence in data and expanding analytical capabilities.

Study Objectives and Overview

This work describes the development and validation of an LC-HRAM method for the simultaneous quantification of 26 AEDs in human plasma or serum. The method employs a Thermo Scientific Vanquish Duo UHPLC system coupled to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer. Unlike conventional triple-quadrupole assays, this HRAM approach leverages full-scan acquisition with data-dependent MS2 (ddMS2) for both quantitation and confirmation of analytes, supporting routine clinical research and in-depth qualitative investigations.

Methodology and Instrumentation

Sample Preparation

• A 50 µL sample of plasma or serum spiked with internal standards was protein-precipitated using a proprietary ClinMass® kit (RECIPE Chemicals), followed by vortexing, centrifugation, and a 10-fold dilution of the supernatant.
• Four calibrators (including blank), two quality controls, and 23 deuterated internal standards covered target concentration ranges for each compound.

Chromatography

• A Vanquish Duo UHPLC system (LC-only mode) employed a 4.5 min gradient at 0.6 mL/min with mobile phases and analytical column supplied by RECIPE Chemicals.
• Two injections per batch were performed: one in positive-ion mode and one in negative-ion mode.

Mass Spectrometry

• Analysis was conducted on a Q Exactive Plus Orbitrap with heated electrospray ionization (H-ESI II).
• FullMS scans (70,000 resolution at m/z 200) over 50–400 m/z and ddMS2 (17,500 resolution) using inclusion lists and compound-specific collision energies enabled quantification and confirmation.

Key Results and Discussion

• Lower limits of quantification (LLOQs) ranged from 0.020 ng/mL (tiagabine) to 8.33 ng/mL (ethosuximide), with all analytes exhibiting CV <20% at LLOQ.
• Calibration curves demonstrated linear responses with 1/x weighting and biases within ±15% (±20% at LLOQ).
• Carryover was negligible (<0.1% for gabapentine and pregabaline; none for others).
• Accuracy assessed with kit controls showed biases between –8.6% and +9.6%; external proficiency controls confirmed biases between –14.5% and +15.1%.
• Intra-assay precision (n=5) CVs were below 9.3%; inter-assay precision over three days was below 7.9%.

Practical Benefits and Applications

This HRAM method offers:

• Comprehensive coverage of 26 clinically relevant AEDs in a single, rapid assay.
• High selectivity and confidence through accurate-mass detection and ddMS2 confirmation.
• Streamlined offline sample preparation compatible with routine clinical research workflows.
• Potential to support both quantitative therapeutic drug monitoring and qualitative metabolite or impurity profiling.

Future Trends and Potential Uses

• Integration of online SPE for further automation and throughput enhancement.
• Expansion to additional neuroactive compounds, metabolites, or biomarkers in epilepsy research.
• Application of artificial intelligence–driven data processing for improved peak detection and deconvolution.
• Adoption of HRAM platforms in routine clinical laboratories as costs decrease and familiarity grows.
• Development of multi-analyte panels combining AEDs with co-medications or endogenous markers to support personalized treatment strategies.

Conclusion

A rapid, robust LC-HRAM method using a Vanquish Duo UHPLC and Q Exactive Plus Orbitrap demonstrates excellent sensitivity, accuracy, and precision for quantifying 26 antiepileptic drugs in human plasma or serum. The approach streamlines sample preparation, minimizes carryover, and provides both quantitative and qualitative data in a single workflow, meeting the demands of clinical research and advancing therapeutic drug monitoring capabilities.

Instrumentation

• Vanquish Duo UHPLC system (Thermo Scientific)
• Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer with H-ESI II (Thermo Scientific)
• ClinMass® TDM platform and Antiepileptics Add-On Kit (RECIPE Chemicals + Instruments GmbH)

Reference

1. De Nardi C., Kern K., Peters S. Quantification of antiepileptics in human plasma or serum by LC-HRAM (MS) for clinical research. Thermo Fisher Scientific Technical Note TN73332-EN. 2020.