Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
47, Number 6, July 2009, pp. 478-484
Development and Validation of a Rapid Column-Switching
High-Performance Liquid Chromatographic Method for the Determination
of Lamotrigine in Human Serum
María del Rosario Brunetto1, Yaritza
Contreras1,
Yelitza Delgado1, Máximo Gallignani1, José Manuel
Estela2,
and Víctor Cerdà Martin2 1Laboratorio de Espectroscopia Molecular Departamento de Química,
Facultad de Ciencias, Universidad de Los Andes, Apartado Postal 440, Mérida,
5101-A, Venezuela; 2Departamento de Química, Facultad de Ciencias,
Universidad de Islas Baleares, Carretera de Valldemossa km 7.5, Palma
de Mallorca, Islas Baleares, España
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.
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