Simultaneous determination of amlodipine besylate and azilsartan mixture in human plasma utilizing high-performance thin-layer chromatography with ultraviolet detection

A novel, efficient, and sensitive high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the concurrent measurement of amlodipine besylate (AML besylate) and azilsartan (AZL) in human plasma spiked with the mixture. Reflectance/absorbance densitometry was conducted using toluene‒ethyl acetate‒methanol‒acetone‒acetic acid (6:1.5:1:0.5:1, V/V) as the mobile phase, and separation was achieved on a precoated silica gel HPTLC plate. This chromatographic system yielded compact bands with excellent resolution at a retardation factor (R_F) of 0.22 ± 0.002 for AML besylate and 0.73 ± 0.001 for AZL. Quantification of AML besylate and AZL was performed at 244 nm within the ranges of 60‒600 ng per band and 90‒900 ng per band, respectively. Calibration plots exhibited strong linearity, with correlation coefficients of 0.9976 for AML besylate and 0.9974 for AZL. Following the International Council for Harmonisation (ICH) guidelines, the developed method was validated. The lowest detectable values for AML besylate and AZL were 13.79 ng per band and 18.62 ng per band, respectively. The recommended HPTLC methodology for the simultaneous determination of AML besylate and AZL is demonstrated to be sensitive, selective, accurate, and precise. This technique can effectively be applied to the simultaneous detection and quantification of AML besylate and AZL in synthetic mixtures and human plasma samples. The enhancing effect of ammonia on the absorption intensity and the bathochromic effect on the wavelength of absorbtion were investigated by molecular modeling and it is suggested that ammonia causes acrylamide to change into acrylamic acid with more conjugated double bonds that rationale the increase in the absorption intensity and the bathochromic shift in the wavelength of the absorption.