Determination of losartan, telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection

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 mu L of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solidphase extraction procedure involving a precolumn packed with 25 mu m C-18 alkyl-diol support (ADS), and a solution 2% methanol in 5 mM 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 5 mM 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 nnn 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 mu g mL(-1) with excellent coefficient ofcletermination (>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 mu g mL(-1) when a sample volume of 20 mu L 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. (C) 2009 Elsevier B.V. All rights reserved.