RESUMO
This study describes a simple and sensitive column-switching high-performance liquid chromatographic method with UV detection for the determination of Lamotrigine in 50 microL of serum. After solid-phase extraction of Lamotrigine on an Oasis HLB extraction precolumn (20 x 3.9 mm; dp: 25 microm), chromatographic separation was achieved at 30 degrees C on a Chromolith RP-18e column (50 mm x 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 microg/mL when a sample volume of 50 microL was injected. However, it was possible to enhance the sensitivity further by injecting larger volumes, up to 200 microL. 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.
Assuntos
Anticonvulsivantes/sangue , Cromatografia Líquida de Alta Pressão/métodos , Triazinas/sangue , Humanos , LamotriginaRESUMO
Column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of losartan, telmisartan, and valsartan in human urine. Urine samples were diluted on the extraction mobile phase (1:4, v/v) and a volume of 20 microL of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solid-phase extraction procedure involving a precolumn packed with 25 microm C(18) alkyl-diol support (ADS), and a solution 2% methanol in 5mM phosphate buffer (pH 3.8) at a flow-rate of 0.8 mL/min for isolation and preconcentration of losartan, telmisartan, and valsartan. The enriched analytes were back-flushed after, onto the analytical column with a mixture of 5mM phosphate buffer (pH 3.8)-acetonitrile-methanol (65:20:15, v/v/v) at a flow-rate of 3.0 mL/min and detected by fluorescence at 259 and 399 nm as excitation and emission wavelength respectively. The separation of losartan, telmisartan, and valsartan was achieved on a Chromolith RP-18e monolithic column. The method provides extraction recoveries from spiked urine samples greater than 93%. Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was <3.5 for all compounds and the inter-day-assay C.V. was < 3.7%. The estimated calibration range was 0.001-2.5 microg/mL(-1) with excellent coefficient of determination (>0.9981). The detection limits for losartan, telmisartan, and valsartan at a signal-to-noise ratio of 5:1 were 0.002, 0.0002 and 0.001 microg/mL(-1) when a sample volume of 20 microL was injected. The proposed method permitted the simultaneous determination of losartan, telmisartan, and valsartan in 8 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to 12 samples/h. The developed column-switching method was successfully applied for the determination of these analytes in human urine samples of patients submitted at ARA-IIs therapy.