Simultaneous LC/MS/MS Quantitation of 20 Antiepileptic Drugs in Human Serum

Significance of the Topic

Therapeutic monitoring of antiepileptic drugs is critical for optimizing patient treatment, avoiding toxicity, and ensuring efficacy. The chemical diversity and concentration ranges of these drugs in serum demand a sensitive, specific, and high-throughput analytical method.

Objectives and Study Overview

This application note describes the development and validation of a single-run LC/MS/MS assay capable of quantifying 20 antiepileptic compounds in human serum. The aim was to achieve wide dynamic range, reliable accuracy, and robust reproducibility to support clinical research and laboratory quality control.

Methodology and Used Instrumentation

Samples were prepared by protein precipitation with methanol, dilution, and centrifugation, followed by a 4 µL injection. Chromatographic separation used an Agilent Poroshell 120 EC-C18 guard (2.1 × 5 mm) and analytical (2.1 × 100 mm) columns at 50 °C. The mobile phases were 2 mM ammonium acetate in water (A) and methanol (B), delivered at 0.4 mL/min with a gradient from 10 % to 95 % B over 7.5 minutes.

Mass spectrometric detection employed an Agilent 6460 Triple Quadrupole with Jet Stream electrospray in dynamic MRM mode, monitoring two transitions per analyte and a phospholipid marker. Both positive and negative ion modes were used to cover the drug panel.

Main Results and Discussion

Calibration curves spanned 12 ng/mL to 200 µg/mL, with top concentrations adjusted per analyte. All correlation coefficients (R²) exceeded 0.997. Accuracy at the lowest calibration level was within 20 % of nominal, and within 15 % at higher levels. Intra- and inter-day precision (CV) remained below 15 % for all compounds, with most below 10 %. Representative chromatograms demonstrated clear separation of all 20 drugs within a 7.5 minute runtime, and isotopically labeled internal standards effectively compensated for matrix effects.

Benefits and Practical Applications of the Method

• Simultaneous quantitation of a broad panel of antiepileptic drugs in a single injection.
• High sensitivity and specificity across multiple orders of magnitude.
• Rapid turnaround time suitable for clinical research and routine monitoring.
• Robustness against serum matrix interferences using internal standards.

Future Trends and Applications

Further work may explore automation of sample preparation, expansion to additional anticonvulsant or co-medication classes, and investigation of alternative sample matrices such as plasma or dried blood spots. Advances in high-resolution MS and microflow LC could improve sensitivity and reduce solvent consumption.

Conclusion

A validated LC/MS/MS method has been established for the reliable quantitation of 20 antiepileptic drugs in human serum, delivering excellent accuracy, precision, and throughput. This approach supports clinical decision-making and quality control in pharmacotherapy management.