Authors :
Presenting Author: Chun-Yi Jimmy Wu, PhD – University of California, Davis
Dorota Zolkowska, MD, PhD. – University of California, Davis
Chris Rundfeldt, PD Dr. med, vet. habil. – PrevEp, Inc.
Pavel Klein, MD – Mid Atlantic Epilepsy and Sleep Center
Michael A. Rogawski, MD, PhD. – University of California, Davis
Rationale:
An intravenous formulation of the antiseizure medication topiramate using meglumine as solubilizing agent is being developed for various applications, including the treatment of seizure emergencies such as pharmacoresistant status epilepticus and neonatal seizures. To support its development and GMP manufacturing, we designed and validated two LC-MS/MS methods, one for the assurance of the topiramate potency and stability in the aqueous topiramate-meglumine formulation and one for determining topiramate concentrations in plasma samples from rats dosed with the formulation in pharmacokinetic and toxicology studies.Methods:
LC-MS/MS method was developed and validated for quantifying topiramate and its related degradants. Chromatographic separation was achieved using a Waters UPLC with a BEH C18 column, utilizing a gradient elution program. Detection was performed using multiple reaction monitoring (MRM) with a Waters Xevo TQ-S mass spectrometer, targeting topiramate (m/z 338 → 78); its internal standard (IS), D12-topiramate (m/z 350 → 78); and potential metabolite, 4,5-desisopropylidene topiramate (m/z 298 → 78). Single ion recording (SIR) was used for monitoring a topiramate impurity A (TPMA) with a sodium adduct, [M+Na]+=283. Formulation samples were spiked with IS in acetonitrile (ACN), diluted with H2O and subjected to LC-MS/MS analysis. Plasma sample preparation involved protein precipitation with IS in ACN, followed by centrifugation and dilution before LC-MS/MS analysis. The method for each sample type was validated for key parameters including linearity, sensitivity, precision, accuracy, extraction efficiency, matrix effect, and stability.
Results:
The validated method exhibited excellent linearity across the tested concentration ranges with correlation coefficients (R²) exceeding 0.99 for all analytes. Intra-batch and inter-batch precision and accuracy were within acceptable limits, with %CV values below 15% and accuracy deviations within ±15%. The method demonstrated high extraction efficiency, with minimal matrix effects observed for all compounds except TPMA, which has 80% extraction efficiency from the formulation probably due to about 20% of inhibitory matrix effect from the formulation. The formulation assay method was successfully applied to confirm the potency of topiramate in a meglumine solution with 20 mg/mL nominal dissolved topiramate. The plasma topiramate assay method was successfully applied to a toxicokinetic study in rats, which revealed significant sex-based differences in pharmacokinetics, particularly in half-life and clearance rates, with females showing longer half-life and lower clearance compared to males.
Conclusions:
The LC-MS/MS methods offer reliable and precise quantification of topiramate and its potential degradants and metabolites in the formulation and in biological matrices. The methods enable manufacturing of the topiramate formulation, and pharmacokinetic and toxicokinetic studies. The observed sex differences in topiramate pharmacokinetics in rats highlights the importance of considering gender in dosing of topiramate, particularly when used in critical conditions like seizure emergencies.
Funding:
Supported by NINDS grant R43NS132659