**1. Introduction**

The consumption of sprouts, common in Asia, has been growing in western countries in the last decade once they were found to possess a broad spectrum of biologically active properties such as antioxidant, anti-inflammatory, allelopathic, and viewed as a valuable dietary supplement [1–4]. *Medicago sativa L* and *Trifolium pratense* sprouts are commonly consumed worldwide. They belong to the *Fabaceae* family, generally known by their commonly edible seeds [4–6].

Due to the increase in the use of sprouts in the human diet, there has been an expansion in the scientific literature regarding their phytochemical contents and biological proprieties. Nutritional properties of *Fabaceae* have been investigated in numerous studies [1,7].

The most interesting secondary metabolite classes found in *Medicago species* are the triterpene saponins and the polyphenolic compounds. The extraction, profiling, and identification of *M. sativa* saponins were extensively studied [8–11]. In contrast to the saponins, *Medicago* polyphenols are less genus-specific and generally encountered in many legumes. Nonetheless, alfalfa contains specific potentially valuable flavonoids with phyto-estrogenic abilities, which makes it particularly interesting for chemical characterization and pharmacological studies [12].

Red clover (*Trifolium pratense* L) sprouts are recognised as a source of phytoestrogens with high biological activity and as a dietary supplement reducing menopausal symptoms [13,14]. However, most studies focused only on certain classes of phenolic compounds such as isoflavones compounds with phytoestrogenic activity and their glycosides [13,15,16]. Among them, other polyphenolic compounds were identified by high-performance liquid chromatography HPLC in *Trifolium pratense L*.: glycitein, pratensein, pseudobaptigenin, and prunetin [16]. The comprehensive profile of phenolic compounds in aerial parts of *Trifolium pratense L*. extracts was obtained by the HPLC-tandem mass spectrometry HPLC-MS/MS technique [17,18].

Fewer reports have developed methods for qualitative and/or quantitative analysis of polyphenols in alfalfa. Flavonoids content in alfalfa was analysed in aerial parts [19,20] or in commercial sprouts by HPLC [5,6]. Ferulic acid, luteolin, myricetin, and *p*-cumaric acid were quantified in alfalfa sprouts by Oh and Rajashekar (2009) [1] using an HPLC system. However, due to the limitation of applied instrument methods, only high-level components were studied in previous studies. Moreover, a comprehensive overview of the polyphenols content variation in red clover and alfalfa during germination has not yet been reported.

High resolution mass spectrometry (HRMS), which is able to provide the accurate mass of unknown compounds, has become an important tool for characterizing chemical components in a natural product [21,22]. In the present work, we describe a comparative study conducted on alfalfa and red clover seeds and sprouts during different germination stages. Extensive characterisation of polyphenolic compounds was done by quantitative and qualitative analysis performed using ultra high-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS). Thus, 29 compounds were tentatively identified without a reference standard, based on their retention times, mass spectra in a full scan mode (MS), and fragmentation patterns observed in MS-MS mass spectra. A number of 30 major compounds were unambiguously identified and quantified by comparing with reference standards.

A fully non-targeted approach of data acquisition with and without fragmentation in one single run was developed. A full scan acquisition event without fragmentation at 70,000 full width at half-maximum FWHM of resolving power was followed by five consecutive fragmentation events at a resolving power of 35,000 FWHM (variable data independent acquisition, *v*DIA) where all ions from the full scan range are fragmented. Data Independent Acquisition is an advanced option to perform untargeted fragmentation, where the entire full scan mass range is segmented in a number of subsequent fixed m/z precursor ion ranges, which are fragmented subsequently. Thus, the fragment can be restricted to the masses in a certain fragmentation event. Zomer and Mol (2015) [23] and Elmiger (2018) [24] used this approach in the analyses of small molecules as pesticides and drugs. Compared to all-ion fragmentation, vDIA can improve selectivity because product ions result from a smaller range of precursor ions [23,24].

The data obtained were subjected to statistical processing using multivariate analysis (PCA) and hierarchical clustering analysis (HCA). The present study results could represent a novel opportunity for food science and health promotion so that only certain classes of phenolic compounds in alfalfa and red clover plants/sprouts were described in previous works [13,15,18,19]. Based on our knowledge, this is the first comparison study on the chemical profile of polyphenolic compounds in sprouts of red clover and alfalfa in different germination stages.
