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 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.
del Rosario Brunetto
María
M
Laboratorio de Espectroscopia Molecular, Departamento de Química, Facultad de Ciencias, Universidad de Los Andes, Apartado Postal 440, Mérida 5101-A, Venezuela. brunetto@ula.ve
Contreras
Yaritza
Y
Clavijo
Sabrina
S
Torres
Dina
D
Delgado
Yelitza
Y
Ovalles
Fernando
F
Ayala
Carlos
C
Gallignani
Máximo
M
Estela
José Manuel
JM
Martin
Víctor Cerdà
VC
D000959:Antihypertensive Agents / Q000652:urine*; D001562:Benzimidazoles / Q000652:urine*; D001565:Benzoates / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D019808:Losartan / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000077333:Telmisartan; D013777:Tetrazoles / Q000652:urine*; D014633:Valine / Q000031:analogs & derivatives* / Q000652:urine; D000068756:Valsartan
D000959:Antihypertensive Agents; D001562:Benzimidazoles; D001565:Benzoates; D013777:Tetrazoles; D000068756:Valsartan; D014633:Valine; D019808:Losartan; D000077333:Telmisartan