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Fast HPLC-UV method for the simultaneous determination of carbamazepine, lamotrigine, oxcarbazepine and phenytoin in mouse plasma, brain and liver tissues Introduction : With a prevalence of about 0.6-0.8%, epilepsy is one of the most common serious chronic neurological disorders affecting people worldwide (1). Although seizures are usually controlled with oral pharmacological medication, up to 30% of epileptic patients are refractory even when prescribed with one or more antiepileptic drugs (AEDs). Furthermore, the polytherapy with AEDs may lead to unpredictable pharmacokinetic interactions and toxicity. In order to maximize the therapeutic benefits of AEDs combinations, appropriate AED polytherapy regimens must be investigated and the most promising dose combination must be defined. In vivo non-clinical isobolographic analysis has demonstrated to be a useful tool to provide a valuable insight towards a rational polytherapy. However, appropriate analytical methodology must be available to support that analysis. Aim : The present study aimed to develop and validate a simple and reliable high-performance liquid chromatographic method (HPLC) with ultraviolet (UV) detection for the simultaneous quantitative determination of carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenytoin (PHT) and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), licarbazepine (Lic) and 5-(4-hydroxyphenyl)-5-phenylhydantoin in mouse plasma, brain and liver. Materials and Methods : Blank samples of plasma (200 µL), brain (500 µL) and liver (250 µL) homogenate supernatants from male CD-1 mice were spiked with known amounts of all analytes and internal standard (IS, LY-83583). All samples were processed applying a solid-phase extraction procedure using Waters Oasis® HLB cartridges. However, plasma and liver tissue homogenates were previously submitted to a deproteinization with acetonitrile. Chromatographic separation was achieved on a LiChroCART Purospher® Star (C18, 3 µm, 55 mm x 4 mm) column using an isocratic elution at 1.2 mL/min with a mobile phase composed by water, methanol, acetonitrile and triethylamine (68.7:26:5:0.3, v/v/v/v) at pH=7.5 adjusted with ortho-phosphoric acid (85%). The selected wavelength of detection was 240 nm for plasma and liver and 237 nm for brain sample analysis. The described assay was validated according to the Food and Drug Administration validation guideline. Results : Chromatographic separation was attained in less than 13 min and no significant matrix endogenous interferences were observed at the retention time of the analytes and IS. Calibration curves were linear with regression coefficients higher than 0.991. The overall inter- and intra-day precision (% CV) did not exceed 15% and the accuracy (% bias) was within ±15%. The lower limit of quantification (LLOQ) established for all the compounds, in plasma, ranged from 0.1 to 1 µg/mL, while in brain and liver tissues was between 0.05-0.2 µg/mL and 0.2-0.5 µg/mL, respectively. The precision and accuracy at LLOQ were also demonstrated and did not exceed the absolute value of 20%. The compounds were efficiently extracted from all the matrices, presenting recoveries greater than 87.1%. Conclusions : A sensitive, accurate and reliable HPLC-UV method for the simultaneous quantification of the considered AEDs in mouse plasma, brain and liver tissues was successfully developed and validated. This assay seems to be a suitable tool to support preclinical pharmacokinetic studies in order to optimize the combination therapy of such AEDs, improving their therapeutic efficacy and tolerability. References : 1. Landmark CJ, Johannessen SI and Tomson T. DOI: 10.1016/j.addr.2011.10.003 Acknowledgements : Fundação para a Ciência e a Tecnologia (SFRH/BD/64895/2009).
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