Quantitative LC-MS/MS Method for the Simultaneous Measurement of Six Antiepileptics and Pentobarbital in Human Serum

Antiepileptic drugs (AEDs) are a chemically diverse group of medications that are used to control seizures and different clinical forms of epilepsy. AEDs can be used as single agents but are commonly administered in combination, as a multi-drug regimen. AEDs have narrow therapeutic windows. Therapeutic ranges may not be properly defined, and symptoms of toxic serum concentrations may include increased frequency of seizures, as seen when AED concentrations are subtherapeutic. Pentobarbital, a barbiturate, is a potent anti-seizure medication, but it is also used in the treatment of head injury. Therapeutic drug monitoring (TDM) is required for optimal treatment of epilepsy. The method presented here is designed to measure serum concentrations of six commonly administered antiepileptic drugs (levetiracetam (Keppra), lamotrigine, lacosamide, 10-hydroxycarbazepine (oxcarbazepine metabolite), topiramate, zonisamide) and that of pentobarbital by LC-MS/MS. Liquid-liquid sample extraction is followed by reversed-phase chromatography using biphenyl HPLC column and gradient elution. Two MRM transitions are monitored for each drug, and their heavy isotope labeled internal standards. Six-point calibration curve is generated with each batch of analysis for quantitation of AEDs. The method's AMR covers the clinically relevant concentration range for each AED. The method has <10% CV throughout the AMR, is free of matrix effect commonly found in clinical samples, and is free from cross reactivity by other AEDs.