Identification of Chlorogenic Acid from Morus alba Leaves by UV-Vis Spectroscopy, FTIR, UPLC-QTOF-MS and Quantification by HPTLC

Abstract

ABSTRACT The present study aims to characterize chlorogenic acid extracted from Morus alba leaves by using suitable methods such as UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and further quantification by high-performance thin layer chromatography (HPTLC). Sonication method and methanol solvent (50:50%, v/v) is found more effective for the extraction of chlorogenic acid from leaf extract. UV-Vis spectra has shown maximum absorbance at λmax 330 nm, and found overlapping with standard chlorogenic acid. The functional groups of chlorogenic acid were identified by FTIR which indicated the presence of hydroxyl, carboxyl and carbonyl groups. In UPLC-QTOF-MS study, the mass spectra and total ion chromatogram analysis revealed the presence of structural isomers for chlorogenic acid at same m/z of parent ion 353. Mass fragments of daughter ions indicated base peak at m/z 191, m/z 173 and 179 were assigned as 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 3-O-caffeoylquinic acid. Quantitative analysis of chlorogenic acid was performed by HPLTC, overlapping absorption spectra of plant extract revealed great consistency with standard, confirms the specificity of HPTLC method. Similar retention factor (Rf) at 0.14 confirms the presence of chlorogenic acid in leaf extract. The standard plot shows good linear relationship from 200 to 1000 ng/spot (R2 = 0.9986). The detection limit was 15.45 ng/spot while limit of quantification was 46.84 ng/spot, with good average recovery rate between 97.96–99.03%. The amount of chlorogenic acid was quantified 10.8 mg/g dried weight extract.