Regresar

Development and validation of a rapid column-switching high-performance liquid chromatographic method for the determination of Lamotrigine in human serum

Abstract:

This study describes a simple and sensitive column-switching high-performance liquid chromatographic method with UV detection for the determination of Lamotrigine in 50 μL of serum. After solid-phase extraction of Lamotrigine on an Oasis HLB extraction precolumn (20 × 3.9 mm; dp: 25 μm), chromatographic separation was achieved at 30°C on a Chromolith RP-18e column (50 mm × 4.6 mm i.d.) using a solution of 20% acetonitrile in 15 mM phosphate buffer (pH 7.0) as the mobile phase, at a flow-rate of 2.0 mL/min. The eluant was detected at 215 nm. The retention time for Lamotrigine was 1.28 min. The total analysis time was ca. 5 min. However, the overlap of sample preparation, analysis, and reconditioning of the precolumn increased the overall sample throughput to one injection every 3 min. The method was validated for system suitability, linearity, precision, accuracy, robustness, and limit of quantitation. The linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.9996. Recovery studies achieved from Lamotrigine spiked plasma samples showed values greater than 93%, demonstrating the excellent extraction efficiency of the precolumn. Intra- and inter-day precision were generally acceptable; the coefficient of variation was < 2.3% in all cases. The detection limits for Lamotrigine at a signal-to-noise ratio of 3 was 0.002 μg/mL when a sample volume of 50 μL was injected. However, it was possible to enhance the sensitivity further by injecting larger volumes, up to 200 μL. The method was shown to be robust and the results were within the acceptable range. The method was successfully applied to the determination of Lamotrigine in human serum samples of patients submitted to Lamotrigine therapy.