Plant metabolites, while looking through HRMS: Characterization of the phenolic profile of Lactuca sativa as a case study

Abstract

Higher plants synthesize a wide range of secondary metabolites, such as phenolics, terpenoids and alkaloids, which contribute to odour, taste and colour to the plants and are involved in protection mechanisms against biotic and abiotic stresses. These compounds also have an important role in the food, pharmaceutical and cosmetic industries, since they are use as antimicrobials, antioxidants, colouring-agents, flavouring-agents, agrochemicals, biopesticides, among others. In particular, most of these plant-derived compounds relevant for industrial applications are phenolics (e.g. phenolic acids, flavonoids, coumarins, and lignans). For the investigation of structure-activity relationships and quality control, the access to rapid and reliable analytical methods for the identification and determination of these natural compounds is important. Mass spectrometry coupled to liquid chromatography and UV-visible detection is a powerful tool for the analysis of phenolic compounds. Technological advances introduced in the last years have provided improvements in terms of chromatographic separation but particularly in the field of mass spectrometry. Indeed, the emergence of ultrahigh performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS), such as the time-of-flight mass spectrometry (ToF/MS) or a quadrupole-ToF (QToF/MS) instruments, made possible to develop a very attractive analytical methods that allow to perform high resolution and accurate mass measurements of the precursor and fragment ions, providing valuable structural information for irrefutable compound identification.This Chapter aims to present a powerful analytical methodology for the characterization of phenolic compounds in complex plant material. The phenolic profiles of different lettuce cultivars (butterhead, green oak-leaf and red oak-leaf) were thoroughly studied since this leafy vegetable is one of the most popular in the world, constituting a major dietary source of phenolic compounds. The analytical strategy included the use of UHPLC coupled online to diode array detection (DAD), electrospray ionization interface (ESI) and QToF/MS. MSE acquisition mode was also used to maximize the QToF instrument duty cycle by collecting automatic and simultaneous information of exact mass at high and low collision energies of precursor ions, as well as other ions produced as a result of their fragmentation, over a single experimental run. One hundred seventeen phenolic compounds were identified in the acidified hydromethanolic extract of freeze-dried lettuce leaves: 40 hydroxycinnamic acid derivatives, 21 hydroxybenzoic acid derivatives, 2 hydroxyphenylacetic acid derivatives, 18 flavonols, 11 flavones, one flavanone, 4 anthocyanidins, 7 coumarins, one hydrolysable tannin and 12 lignans. Forty eight of these compounds were tentatively identified for the first time in lettuce in the present study, and only 20 of them had been previously reported in oak-leaf lettuce cultivars in literature. Moreover, the phenolic profile of the butterhead lettuce cultivar had not been described before. The UHPLC-DAD-ESI-QToF/MSE approach provided new structural information and allowed the identification of unknown phenolics demonstrating to be a useful tool for the characterization of phenolic compounds in complex plant matrices.