Phenytoin (PHT), phenobarbital (PHB), lamotrigine (LTG), and topiramate (TPM) are some of the most widely used antiepileptic drugs (AEDs). Monitoring of their concentrations in serum is important for the treatment of epilepsy. A reference measurement procedure (RMP) for certification of PHT, PHB, LTG, and TPM in serum has been developed and critically evaluated. Isotopically labeled compounds of PHT, PHB, LTG, and TPM are used as internal standards for the four AEDs. The four drugs and their respective labeled internal standards are simultaneously extracted from serum using solid-phase extraction prior to reversed-phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a C18 column. Electrospray ionization (ESI) in the positive ion mode for PHT and LTG, and in the negative ion mode for PHB and TPM were used. The recovery of AEDs added to serum (accuracy of the extraction method) was evaluated by recovery studies of measuring the four drugs in spiked samples with known drug levels. The recoveries of the added drugs ranged from 98.6% to 102.0%. The absolute recoveries (extraction efficiencies) of the four drugs with this method ranged from 97% to 100%. Excellent repeatability was obtained for the four drugs with between-set coefficients of variation (CVs) within 1%. The type B components estimates are conservatively large and are considerably larger than the type A component. Therefore, we use the usual metrological expansion factor of 2 to provide an approximate 95% coverage interval. The relative expanded uncertainties for the four AEDs ranged from 2.3% to 2.4%. This LC-MS/MS RMP for PHT, PHB, LTG, and TPM in serum demonstrating good accuracy and precision can be used to assess the accuracy of routine methods used in clinical laboratories.
