Qualitative and Quantitative Mass Spectrometry Approaches for the Analysis of Phenolic Compounds in Complex Natural Matrices

Phenolic molecules represent one of the most prevalent and biologically important categories of secondary metabolites. Within this diverse group, phenolic acids and flavonoids are the most extensively studied categories, primarily due to their structural diversity and broad spectrum of reported bioactivities. We first provide an overview of the physicochemical characteristics of flavonoids and phenolic acids and discuss how these properties relate to mass spectrometry (MS) fragmentation patterns and chromatographic behavior, including retention characteristics and isomer resolution. Next, we systematically examine the utilization of MS-based procedures for the characterization of flavonoids and phenolic acids in complex natural matrices. We initially examine targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing triple-quadrupole (QQQ) platforms, focusing on selected/multiple reaction monitoring (SRM/MRM) and associated scanning techniques (product-ion and precursor-ion scans). We summarize validated methodologies and strategies for both absolute and relative quantification, including stable-isotope dilution, matrix-matched calibration or standard addition, and internal-standard normalization. We subsequently analyze untargeted high-resolution mass spectrometry methodologies (direct injection and coupled to liquid chromatography), highlighting recent progress in data acquisition while addressing ongoing challenges in computational processing. Finally, we present a brief evaluation of commonly used extraction and clean-up processes, highlighting their practical impact on phenolic recoveries.