Development and validation of a liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation to determine levetiracetam in epileptic patient serum and its application in therapeutic drug monitoring.

Abstract

Levetiracetam is an antiepileptic drug that is primarily approved by the Food and Drug Administration for the treatment of focal and generalized seizures. This study describes the development and validation of a highly selective and sensitive liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation to determine levetiracetam in epileptic patient serum. After simple protein precipitation, the analytes were separated on a short reversed-phase column (Agilent Poroshell 120 SB-C18 column, 4.6 × 50mm, 2.7μm) using isocratic elution with 25% 0.1% formic acid in water (solvent A) and 75% methanol (solvent B) at a flow rate of 0.8ml/min. The linear range is 0.5-50 μg/mL (R2 >0.99). All the validation data, such as lower limit of quantification, linearity, specificity, recoveries, matrix effects, and other parameters, fit the request of biological method validation guidance. Passing-Bablok regression coefficients demonstrated that there is no constant bias and no proportional bias between the liquid chromatography-tandem mass spectrometry methods with triple-stage fragmentation and liquid multiple reaction monitoring. Bland-Altman plot showed that the developed liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation method is reliable and accurate to determine levetiracetam in human serum.