Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry

BackgroundLevetiracetam (Keppra) is a novel antiepileptic drug recently approved by the U.S. Food and Drug Administration as an add-on therapy in the treatment of partial onset seizures in patients. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of levetiracetam in human matrix (plasma, serum, or saliva).MethodsAn API-3000 or API-4000 triple–quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the IonSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing ritonavir as internal standard (IS) for levetiracetam. One hundred microliters of serum (or plasma, saliva) was deproteinized by adding 150 μl of acetonitrile containing internal standard. After centrifugation, 100 μl of supernatant was diluted with 300 μl of water and 10 μl aliquot was injected onto a C-18 column. After a 2.5 min wash the valve was activated to initiate the isocratic elution program which eluted the levetiracetam and internal standard into the MS/MS system. Quantitation by MRM analysis was performed in the positive ion mode. Within-day and between-day imprecision were evaluated for levetiracetam using three levels of in-house controls. Reliability and accuracy of this method were assessed by comparison of targets with external QC material (ChromSystems), between laboratory comparisons and by recovery studies.ResultsWithin-day coefficients of variation (CVs) were < 6.1% and between-day CVs were < 8.2%. The average spiked recoveries of levetiracetam added to the drug-free human plasma samples were 108% at low concentration level and 103% at high concentration level.ConclusionsThe method was found both specific and sensitive for the rapid and accurate measurement of levetiracetam in human matrices and correlated well with the Quest/Chantilly tandem mass spectrometric method (r = 0.983).