Abstract
This study describes the development and validation of a highly sensitive high-performance liquid chromatographic method with fluorescence detection and on-line emission wavelength switching for the simultaneous determination of valsartan (VAL) and amlodipine (AML) in human plasma. Irbesartan (IRB) was used as internal standard (IS). VAL, AML, and IRB were isolated from plasma by nonextractive procedure; simple protein precipitation with methanol. Separations were performed in low pressure gradient mode on Hypersil phenyl 120A analytical column using a mobile phase consisting of phosphate buffer (pH 4.0 ± 0.1):acetonitrile:methanol (60:30:10, v/v/v) at a flow rate of 0.8 mL/min. The detection of VAL and IRB (IS) was carried out at 253 nm (for excitation) and 374 nm (for emission). After elution of VAL and IRB, the detection wavelengths were switched on-line to 393 nm (excitation) and 446 nm (emission) for detection of AML. The linear ranges of the assay were 1-100 and 10-1000 ng/mL for VAL and AML, respectively. The limits of detection (LOD) were 0.3 and 1.6 ng/mL for VAL and AML, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.1%. The accuracy of the method was proved; the recovery of VAL and AML from spiked human plasma were 96.6-100.9 and 92.0-104.4% for VAL and AML, respectively. The method had higher throughput as it involved simple sample preparation procedure and short run-time (15 min). The results demonstrated that the proposed method would have great value in pharmacokinetic studies for VAL and AML.