42 A Rapid UPLC-MS/MS Assay for the Simultaneous Measurement of Gabapentin, Lamotrigine, Levetiracetam, Monohydroxy Derivative (MHD) of Oxcarbazepine, and Zonisamide Concentrations in Serum

Abstract Background Worldwide, approximately 50 million people live with epilepsy, a quarter of which are treated with antiepileptic drugs (AEDs). Therapeutic drug monitoring (TDM) of AEDs is often necessary to assess therapeutic compliance, potential drug-drug interactions, and pharmacokinetic inter- and intravariability secondary to significant differences in excretion and metabolism. TDM is necessary to minimize the destructive toxicities of AEDs including hematopoietic dysfunction, pruritus, rash, Steven-Johnson syndrome, anxiety, agitation, suicidal ideation, liver failure, gastrointestinal issues, and neurologic dysfunction. Tandem mass spectrometry with stable isotope internal standards is the gold standard for the measurement of AEDs. Here, we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous measurement of gabapentin, lamotrigine, levetiracetam, monohydroxy derivative (MHD) of oxcarbazepine (the main, active oxcarbazepine metabolite, also known as licarbazepine), and zonisamide in serum. Methods For analysis, 20 µL serum samples were added to acetonitrile in the presence of deuterated internal standards for protein precipitation. After centrifugation, 10 µL of supernatant was added to 90 µL of water. Ten µL of this solution was injected into a C18 column for reversed phase UPLC chromatographic separation. Quantification of analytes was accomplished using positive-mode electrospray ionization and collision-induced dissociation MS. Total run time was 3 minutes. Assay parameters were evaluated according to Food and Drug Administration (FDA) bioanalytical guidelines. Results To guide our assay development, we evaluated the range of AED levels for our patient population from lab results assayed at a reference laboratory. Over the course of one year, 1,825 total samples were assayed. One levetiracetam drug level was greater than 100 ug/mL, and the lower limit of quantification (LLOQ) for all drugs analyzed ranged between 2 ug/mL and 0.2 ug/mL. Therefore, an assay range of 0.1 ug/mL to 100 ig/mL was chosen for all analytes and was linear (r2 > .999) from 0.1 ug/mL to 100 ug/mL. This assay demonstrated acceptable accuracy and precision (%DEV