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Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid-liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites.

Analysis of Nucleosides and Nucleotides in Plants: An Update on Sample Preparation and LC–MS Techniques