title: Bridging the gap between comprehensive extraction protocols in plant metabolomics studies and method validation.
abstract: It is vital to pay much attention to the design of extraction methods developed for plant metabolomics, as any non-extracted or converted metabolites will greatly affect the overall quality of the metabolomics study. Method validation is however often omitted in plant metabolome studies, as the well-established methodologies for classical targeted analyses such as recovery optimization cannot be strictly applied. The aim of the present study is to thoroughly evaluate state-of-the-art comprehensive extraction protocols for plant metabolomics with liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS) by bridging the gap with method validation. Validation of an extraction protocol in untargeted plant metabolomics should ideally be accomplished by validating the protocol for all possible outcomes, i.e. for all secondary metabolites potentially present in the plant. In an effort to approach this ideal validation scenario, two plant matrices were selected based on their wide versatility of phytochemicals: meadowsweet (Filipendula ulmaria) for its polyphenols content, and spicy paprika powder (from the genus Capsicum) for its apolar phytochemicals content (carotenoids, phytosterols, capsaicinoids). These matrices were extracted with comprehensive extraction protocols adapted from literature and analysed with a generic LC-PDA-amMS characterization platform that was previously validated for broad range phytochemical analysis. The performance of the comprehensive sample preparation protocols was assessed based on extraction efficiency, repeatability and intermediate precision and on ionization suppression/enhancement evaluation. The manuscript elaborates on the finding that none of the extraction methods allowed to exhaustively extract the metabolites. Furthermore, it is shown that depending on the extraction conditions enzymatic degradation mechanisms can occur. Investigation of the fractions obtained with the different extraction methods revealed a low resolving power for phytochemicals for all methods. Nevertheless, an overall good repeatability was observed for all extraction methods, which is essential to allow direct comparison between samples. In summary, no single procedure outperforms the others and compromises will have to be made during method selection.
authors.affiliation: University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium. Electronic address: sebastiaan.bijttebier@uantwerpen.be.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Bijttebier
authors.affiliation: University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium. Electronic address: anastasia.vanderauwere@uantwerpen.be.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Van der Auwera
authors.affiliation: University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium. Electronic address: kenn.foubert@uantwerpen.be.
authors.forename: Kenn
authors.initials: K
authors.lastname: Foubert
authors.affiliation: Flemish Institute for Technological Research (VITO), Business Unit Separation and Conversion Technology (SCT), Mol, Belgium. Electronic address: stefan.voorspoels@vito.be.
authors.forename: Stefan
authors.initials: S
authors.lastname: Voorspoels
authors.affiliation: University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium. Electronic address: luc.pieters@uantwerpen.be.
authors.forename: Luc
authors.initials: L
authors.lastname: Pieters
authors.affiliation: University of Antwerp, Natural Products & Food Research and Analysis (NatuRA), Antwerp, Belgium. Electronic address: sandra.apers@uantwerpen.be.
authors.forename: Sandra
authors.initials: S
authors.lastname: Apers
mesh_terms: D002212:Capsicum / Q000737:chemistry* / Q000378:metabolism; D031984:Filipendula / Q000737:chemistry* / Q000378:metabolism; D055432:Metabolomics; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010936:Plant Extracts
keywords: Comprehensive phytochemical extraction; Liquid-chromatography-accurate mass mass spectrometry; Plant metabolomics; Validation