A Multiplex LC-MS/MS Approach for Quantifying Antiepileptic Drugs for Therapeutic Drug Monitoring: Validation of 15 Representative Tests

Abstract

INTRODUCTION: Therapeutic drug monitoring of antiepileptic drugs is critical for optimizing clinical outcomes, minimizing toxicity, assessing drug compliance, and managing overdoses and drug interactions. OBJECTIVES: We developed and validated an LC-MS/MS method for the quantification of 15 antiepileptic drugs (ethosuximide, primidone, pentobarbital, carbamazepine-10,11 epoxide, pregabalin, gabapentin, zonisamide, lacosamide, rufinamide, felbamate, lamotrigine, topiramate, 10,11-dihydro-10-hydroxycarbamazepine, perampanel, and brivaracetam). METHODS: Antiepileptic drugs were extracted from plasma and serum using methanol-based protein precipitation, followed by dilution. A commercial ClinCal^® 3-point calibrator was used for all analytes except pentobarbital, which used a 6-point in-house calibrator. Chromatographic separation was achieved using a reverse-phase C18 column with a 7.31 min elution gradient of water and methanol, both containing 2 mM of ammonium acetate. RESULTS: All analytes required a 5 µL injection volume, except ethosuximide and pentobarbital, which required 20 µL and 10 µL, respectively, for optimal performance. The method demonstrated excellent linearity for all 15 antiepileptic drugs within their respective concentration ranges, with acceptable selectivity, accuracy (80.0–118.3%), and intraday and interassay precision (CV < 7.4%). Stability studies showed that antiepileptic drugs were stable in serum and plasma for up to 24 h at room temperature, 7 days at 4 °C, and 30 days at −20 °C, and after extraction for 7 days at 4 °C. CONCLUSION: This LC-MS/MS method supports routine quantification of 15 antiepileptic drugs in a clinical laboratory using a common sample preparation and chromatography workflow across all measurements. It was operationalized into three instrument methods to accommodate the different injection volume and calibrator requirements.