Determinação de compostos fenólicos como marcadores analíticos em extratos de plantas medicinais da Amazônia por UHPLC-ESI-MS/MS

Abstract

A method for separation, identification and quantification of 24 phenolic compounds using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLCMS/ MS) was developed and validated. Six plant species originating from the Brazilian Amazon region and traditionally used as medicinal plants by local people were studied (Mansoa alliacea, Bauhinia variegata var. variegata, Bauhinia variegata var. alboflava, Connarus perrottetii var. angustifolius, Cecropia obtusa and Cecropia palmata). The analytes were separated by a reversed phase SB-C18 column (2.1 × 50 mm, 1.8 μm) using a gradient elution composed of 0.1% acetic acid in water (v/v) and acetonitrile, at a constant flow rate of 0.8 mL min-1. The limit of detection ranged between 0.5 and 130.3 μg kg-1. Intra- and inter-day precision showed satisfactory results and the recoveries obtained for the 24 analytes varied between 91.7 and 123.8% for the evaluated matrices. Moreover, a systematic study on the various sources of ion suppression in UHPLC-MS/MS analysis was carried out for the 24 phenolic compounds in extracts of all the six medicinal plants. The contributions of matrix effects, mobile-phase additives, analyte co-elution and electric charge competition during ionization to the global ion suppression were evaluated. Herein, the influence of mobile-phase additives on the ionization efficiency was found to be very pronounced, where ion suppression of approximately 90% and ion enhancement effects greater than 400% could be observed. The negative effect caused by the wrong choice of internal standard (IS) on quantitative studies was also evaluated and discussed from the perspective of ion suppression. This work also shows the importance of performing studies with this approach even for very similar matrices, such as varieties of medicinal plants from the same species, because different effects were observed for the same analyte. Furthermore, quantification determinations of the 24 analytes were performed with the standard addition method (r > 0.99) using data acquired in selected reaction monitoring (SRM) mode. Rutin, apigenin, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and trans-- cinnamic acid were found in all the studied plants in at least one of the analysed extracts. It should be emphasized the high content of rutin in varieties of Bauhinia variegata (up to 2.52 mg g-1 of plant), chlorogenic acid in species of genus Cecropia (up to 0.57 mg g-1 of plant) and catechin in Connarus perrottetii var. angustifolius (1.77 mg g-1 of plant). Possible analytical markers and potential active markers for quality control of herbal drugs were assigned to each plant species.