High Pressure Liquid Chromatography for Simultaneous Analysis of Anticonvulsants: Comparison with EMIT® System

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Summary:We present a new high pressure liquid chromatography (HPLC) method for the simultaneous analysis of primidone, phenobarbital, phenytoin, and carbamazepine in serum. The chromatographic separation is carried out using an Altex model 110-A pump, a 250 × 4.6 mm column containing 5 μm Spherisorb ODS particles and a variable wavelength ultraviolet detector set at 197 nm. The mobile phase is a mixture of acetonitrile, distilled water, and 1.75 M phosphoric acid (27:72.8:0.2). The flow rate is 1.5 ml/min, and the analysis time is 17 min. A 200 μl aliquot of serum is buffered at pH 5 and extracted with dichloromethane. The extract is evaporated to dryness and dissolved in methanol for chromatographic analysis. Cyclopal is used as the internal standard and quantification is achieved using peak height ratios. This HPLC method is evaluated for precision and accuracy with reference to the EMIT® system. The least-squares regression analysis of comparison data for the drugs shows a favorable correlation. Also, a paired t-test indicates no significant difference for the HPLC and EMIT values for primidone, phenobarbital, phenytoin, and carbamazepine. From this study we conclude that this HPLC method could be successfully used for the simultaneous therapeutic monitoring of the four anticonvulsants.

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