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Publication Citation: Development of a Candidate Reference Measurement Procedure for the Determination of Phenytoin, Phenobarbital, Lamotrigine, and Topiramate in Human Serum Using Isotope-Dilution Liquid Chromotography/Tandem Mass Spectrometry

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Author(s): Susan S. Tai; Karen W. Phinney; Chia-Yi Yeh;
Title: Development of a Candidate Reference Measurement Procedure for the Determination of Phenytoin, Phenobarbital, Lamotrigine, and Topiramate in Human Serum Using Isotope-Dilution Liquid Chromotography/Tandem Mass Spectrometry
Published: August 28, 2011
Abstract: 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 candidate reference measurement procedure (RMP) for the determination 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 SPE prior to reversed-phase liquid chromatography-tandem mass (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 accuracy of the 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 of the four drugs with this method ranged from 97 % to 100 %. The difficulty in getting good repeatability for TPM due to the large mass difference between TPM and its internal standard (TPM-d12) was overcame by shortening its retention time. The peak shape of high nitrogen containing compound of LTG was improved using ammonium acetate as an ion pairing reagent in the mobile phase. Excellent repeatability was obtained for the four drugs with between-set coefficients of variation (CVs) of < 1 %. The limit of detection at a signal-to-noise ratio of ~ 3 was 0.8 pg, 13 pg, 0.1 pg, and 0.2 pg for PHT, PHB, LTG, and TPM, respectively. This candidate LC-MS/MS RMP for the determination of PHT, PHB, LTG, and TPM in serum demonstrating good accuracy and precision can be used to assess the accuracy of test methods used in clinical laboratories.
Citation: Analytical Chemistry
Volume: 40
Pages: pp. 1915 - 1922
Keywords: Phenytoin; PHT; Phenobarbital; PHB; Lamotrigine; LTG; Topiramate; TPM; Reference measurement procedure; RMP; Eletrospray ionization; Isotope dilution; Liquid chromatography/tandem mass spectrometry; LC-MS/MS; solid phase extraction
Research Areas: Organic Analytical Chemistry
DOI: http://dx.doi.org/10.1007/s00216-011-5251-5  (Note: May link to a non-U.S. Government webpage)