LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H]⁺. Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS³ experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula.     

Characterization of Secondary Metabolites in Flowers of Sanguisorba officinalis L. by HPLC‐DAD‐MSn and GC/MS

The investigations reported here focus on an in‐depth characterization of the secondary metabolite profile of Sanguisorba officinalis flowers. For this purpose, fresh flowers were extracted with MeOH/H₂O and EtOH/H₂O and the resulting crude extracts fractionated using CH₂Cl₂, AcOEt, and BuOH. Individual compounds were characterized by high performance liquid chromatography and gas chromatography coupled with mass spectrometric detection (HPLC‐DAD‐MSⁿ and GC/MS). MeOH/H₂O extraction and LC/MSⁿ investigations revealed the occurrence of flavonoid glycosides (quercetin, kaempferol), ellagitannin glycosides and four anthocyanins. Among the latter, two components, i. e., cyanidin‐malonyl‐glucose and cyanidin‐galloyl‐hexose, have not been reported for S. officinalis so far. Furthermore, phenylethylamine was characterized for the first time in Sanguisorba by pH value dependent extraction with CH₂Cl₂. In addition, AcOEt and BuOH extracts were analyzed by GC/MS both prior to and after acid hydrolysis of secondary metabolites. For this purpose, the extracts were treated with 1 n HCl solution (105 °C, 1 h) and derivatized with BSTFA. Analyses revealed the occurrence of several classes of phenolic compounds, such as gallic acid, hydroxybenzoic acid, hydroxycinnamic acid and ellagic acid derivatives. Additionally, the most prominent ursane‐type triterpenoid (ziyu‐glycoside I) from Sanguisorba and its corresponding aglycone isomers were detected and assigned based on their characteristic fragmentation patterns.     

Identification of chemical constituents in extracts and rat plasma from Fructus Aurantii by UPLC-PDA-Q-TOF/MS

Introduction – Fructus Aurantii, as a component of several compound formulae, shows many bioactivities. As is well known, the constituents of traditional Chinese medicines are very complex and multiple constituents are responsible for the therapeutic effect. However, the concrete compounds are difficult to confirm. Therefore, studies on the constituents absorbed into serum and their metabolites are necessary. Objective – To search for the active constituents in Fructus Aurantii. Methodology – An ultra‐performance liquid chromatography–photodiode array detector–quadrupole time‐of‐flight mass spectrometry (UPLC‐PDA‐Q‐TOF/MS) method was established to analyse the aqueous extract in Fructus Aurantii and the constituents absorbed into blood. Compound identification was made by matching the empirical molecular formula with those of the reported compounds and UV spectra, and further elucidated by lower energy CID mass spectra. Results – Twelve flavonoid O‐glycosides were detected, and nine compounds were tentatively identified as polymethoxylated flavones. Six parent compounds were identified and four metabolites were observed in rat plasma, two of which were identified as naringenin glucuronide and hesperetin glucuronide, respectively. Conclusion – The approach developed has proved useful in the study of the active constituents in traditional Chinese medicines. Copyright © 2011 John Wiley & Sons, Ltd.     

THE FLAVONOIDS OF THE DECIDUOUS RHODODENDRON OF NORTH AMERICA (ERICACEAE)

The native Azaleas of North America (Rhododendron: subgenus Pentanthera) produce 58 flavonoids in five aglycone classes: flavonols, dihydroflavonols, flavanones, dihydrochalcones and chalcones. A comparison of the flavonoids of selected samples of these species indicated that the compounds generally occur as species specific ensembles. Species were grouped into “alliances” based on common flavonoid constituents and a phylogenetic treatment of the group was developed. Evolutionary trends of flavonoids within subgenus Pentanthera are not well-defined but appear to be associated with a loss of the ability to synthesize some compounds and a decrease in diversity of glycosides and methoxylated flavonoids. Intraspecific variation in flavonoids was found in Rhododendron canescens, R. alabamense and R. austrinum when these were sampled on a population basis.     

Metabolic Alterations in Two Cirsium Species Identified at Distinct Phenological Stages using UPLC‐QTOF/MS

Introduction

Cirsium chanroenicum and C. setidens are commonly used both in traditional folk medicine and as a food source. The quality of different species of Cirsium at different harvest times is a function of their metabolite composition, which is determined by the phenological stage.

Objective

We sought to determine the differences in the metabolite composition of two species of Cirsium during different phenological stages using ultra‐performance liquid chromatography (UPLC) quadrupole time‐of‐flight (QTOF) mass spectrometry (MS).

Methodology

Cirsium chanroenicum and C. setidens plants were collected at the floral budding and full flowering stages. Metabolic profiles of Cirsium extracts were determined using UPLC‐QTOF/MS to characterise the differences between phenological stages, and the major metabolites were quantified using UPLC‐QTOF/MS‐multiple reaction monitoring (MRM).

Results

At the full flowering stage, the levels of phenolic acids as well as components of the phenylpropanoid pathway were increased. Flavonoids predominated at the full flowering stage in both species. The levels of coumaric acid, kaempferol, and pectolinarigenin differed between the two species of Cirsium. Overall, these results suggest that components of the phenylpropanoid metabolic pathway are upregulated in the full flowering stage in Cirsium, although we did observe some variation between the species.

Conclusion

These results will help elucidate the metabolic pathways related to the different phases of the vegetative cycle, and may help determine the optimal season for the harvest of Cirsium with the highest levels of bioactive compounds. Copyright © 2017 John Wiley & Sons, Ltd.     

Malonyl Isoflavone Glycosides in Soybean Seeds (Glycine max Merrill)

The isoflavone constituents in soybean seeds were investigated, and 9 kinds of isoflavone glycosides were isolated from the hypocotyls of soybean seeds. Three kinds were proved to be malonylated soybean isoflavones named 6″-O-malonyldaidzin, 6″-O-malonylglycitin and 6″-O-malonylgenistin by UV, MS, IR and NMR. The malonylated isoflavone glycosides as major isoflavone constituents in soybean seed were thermally unstable, and were converted into their corresponding isoflavone glycosides. All of the isoflavone components produced intensely undesirable taste effects such as bitter, astringent and dry mouth feeling.     

Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono‐ or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high‐performance liquid chromatography combined with high‐resolution tandem (quadrupole‐time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p‐coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high‐resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

A Comparative Study on Polyphenolic Composition of Berries from the Tibetan Plateau by UPLC‐Q‐Orbitrap MS System

Five traditional medicinal food from the Tibetan plateau including Nitraria tangutorum Bobrov (NT), Hippophae rhamnoides L. (HR), Lycium ruthenicum Murray (LR), Lycium barbarum L. (LB) and Rubus corchorifolius L.f. (RC) are rich in phenolic compounds. However, the detailed studies about the phenolic compounds remain scarce. Therefore, we established a rapid method for the simultaneous identification and quantification of the phenolic compounds from berries via Ultra Performance Liquid Chromatography‐Quadruple‐Orbitrap MS system (UPLC‐Q‐Orbitrap MS). This method was verified from many aspects including detection limit, quantification limit, precision, repeatability, stability, average recovery rate and recovery range, and then was used to analyze the phenolic compounds in these five species of berries. Finally, a total of 21 phenolic compounds were directly identified by comparing the retention time and exact mass, of which 14 compounds were identified by us for the first time in berries from the Tibetan plateau, including one flavonoid aglycone (myricetin), 11 phenolic acids (gallic acid, protocatechuate, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p‐coumaric acid, ferulic acid, 2‐hydroxybenzeneacetic acid and ellagic acid), one flavanol (catechin) and one dihydrochalcone flavonoid (phloretin). Quantitative results showed that rutin, myricetin, quercetin and kaempferol were the main flavonoids. Moreover, a variety of phenolic acid compounds were also detected in most of the berries from the Tibetan plateau. Among these compounds, the contents of protocatechuate and chlorogenic acid were high, and high levels of catechin and phloretin were also detected in these plateau berries.     

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Changes in the profile of flavonoid accumulation in Medicago truncatula leaves during infection with fungal pathogen Phoma medicaginis

Medicago truncatula is a model species for the study of the unique secondary metabolism in legumes. LC/MS/MS analysis was used to identify and profile flavonoid glycoconjugates and free aglycones in leaves of M. truncatula (ecotype R108-1) infected with the fungal pathogen Phoma medicaginis. Use of a high resolution analyzer with a collision induced dissociation tandem mass spectrometer (CID MS/MS) permitted structural elucidation of target secondary metabolites and four new acylated flavone glycosides have been identified. Changes in the phytoalexin medicarpin and its isoflavone precursors were quantitatively monitored at various time points after fungal spore application. Application of spores induced disease symptoms in the leaves of infected plants and resulted in an increase in the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside between one and three days post-infection. Relative concentrations of medicarpin were highest five days post-infection. The rapid increase of these molecules was clearly positively correlated to the infection process as certain of them were absent in uninfected leaves, suggesting that the relative rate of their synthesis is tightly related with the infection process.     

Application of LC/MS systems to structural characterization of flavonoid glycoconjugates

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material. Plant tissues contain thousands of natural products fulfilling different roles in plant physiology and biochemistry. These natural products have various biological activities in respect to plants synthesizing them, in their responses to different environmental stresses and are also active principles of food supplements and pharmaceuticals of plant origin. Flavonoids constitute a large group of phenolic secondary metabolites and are probably produced by all terrestrial plant species. More than 9000 glycoconjugates of flavonoids are presently known in the plant kingdom and more than 50 of them may be present in a single plant. For this reason methods of identification and analysis of this group of compounds are particularly demanded. Due to a high number of metabolites present in plant extracts, the isolation and purification of most compounds in amounts suitable for unambiguous characterization with NMR methods is often impossible. For these reasons elaboration of strategies for sufficiently precise structural characterization of compounds present in mixture samples is currently a primary task. Mass spectrometry, thanks to application of different physical phenomena for ionization, separation and detection of analyzed molecules, became the method of choice among analytical methods applied for identification, structural characterization and quantitative analysis of the natural products. Methods of analysis of differently substituted flavonoids (O- and C-glycosides, differentiation of various oligosaccharidic substituents, detection of acylated compounds) are presented in the paper. A proper application of mass spectrometric methods in well-defined and strictly controlled technical parameters of analysis permits obtaining important structural information. Among others, recording collision induced dissociation mass spectra allows identification of compounds after comparison of the registered MS spectra with these present in the existing databases.     

A new generation of cross‐linkers for selective detection by MALDI MS

We designed a new cross‐linker bearing a CHCA moiety. The use of the CHCA‐tagged cross‐linker JMV 3378 in conjunction with a neutral MALDI matrix α‐cyano‐4‐hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI‐TOF MS of cross‐link containing peptides. Discrimination between modified and non‐modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the α‐cyano‐4‐hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo‐myoglobine as model proteins.     

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC,HPLC, UPLC‐ESI‐QTOF‐MS and LC‐SPE‐NMR Fingerprints Analyses

Introduction

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown.

Objective

The present study aims finding suitable conditions to identify the bioactive compounds from different fractions.

Methodology

Chromatographic fingerprint profiles of different fractions were developed using high‐performance liquid chromatography (HPLC) and then these conditions were transferred to thin‐layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra‐performance liquid chromatography‐quadrupole time of flight‐tandem mass spectrometry (UPLC‐QTOF‐MS) and liquid chromatography‐solid phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy.

Results

The HPLC fingerprints, developed on two coupled Chromolith RP‐18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin‐3‐O‐rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di‐pentoside) were tentatively identified by QTOF‐MS, while NMR confirmed the structure of rutin and nicotiflorin.

Conclusion

The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.     

Discrimination of Different Parts of Saffron by Metabolomic‐Based Ultra‐Performance Liquid Chromatography Coupled with High‐Definition Mass Spectrometry

In this study, the metabolite profiling of three different parts of Crocus sativus L. was measured by using ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐QTof‐MS/MS). Multivariate statistical analysis was used to distinguish among the samples from different parts. A total of 54 compounds were identified in tepals, stigmas and stamens by UPLC‐QTof‐MS/MS. The results stated that chemical characteristics of saffron were obviously diverse in terms of the parts of flower. Through analysis, coniferin and crocin‐2 were special components in stigmas when compared to tepals and stamens. The content of flavonoids was high in tepals when compared with the stigmas. The tepal of saffron may processed as a source of flavonoids in the future. The research provided the basis for the theory that only the stigma can be used as medicine.     

Phytochemical Analysis and Total Antioxidant Capacity of Rhizome,Above‐Ground Vegetative Parts and Flower of Three Iris Species

This study was aimed at investigating the phytochemical composition and antioxidant capacity of rhizomes, above‐ground vegetative parts and flowers of three Iris species: Iris humilis Georgi , Iris pumila L. and Iris variegata L. UHPLC‐Orbitrap MS analysis was used for determination of phytochemical profile. Total pigments, phenolics, flavonoids, soluble sugars and starch content as well as ABTS antioxidant capacity were also determined. In total, 52 phenolics compounds were identified with 9 compounds (derivatives of iriflophenone, apigenin C‐glycosides, luteolin O‐glycoside, isoflavones derivatives of iristectorigenin, dichotomitin, nigracin and irilone) never reported before in Iris spp. Differences in phenolic composition profile, pigments, soluble sugar, starch, total phenolics and flavonoids content and total antioxidant capacity were found among Iris species and different part of plants. Significant correlation between total phenolic content and antioxidant capacity was determined. The obtained results are comparable with those obtained for medical plants. These findings could be useful for fingerprinting characterization of Iris species and estimation of possible use in pharmaceutical industries.     

Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning

The performances of five different ESI sources coupled to a polystyrene–divinylbenzene monolithic column were compared in a series of LC‐ESI‐MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low‐flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest‐quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures – arguably due to an increased number of high intensity precursor ion candidates.     

Crystal structure of β‐glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

The β‐glucosidase TnBgl1A catalyses hydrolysis of O‐linked terminal β‐glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3‐glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure‐function relationships, the crystal structure of the nucleophile mutant TnBgl1A‐E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin‐3‐glucoside (Q3), as well as for the model substrate para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3‐glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid‐3‐glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872–884. © 2016 Wiley Periodicals, Inc.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Flavonoids enantiomer distribution in different parts of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.) and phacelia (Phacelia tanacetifolia Benth.)

Plant material is a rich source of valuable compounds such as flavanones. Their different forms influence bioavailability and biological activity, causing problems with the selection of plant material for specific purposes. The purpose of this research was to determine selected flavanone (eriodictyol, naringenin, liquiritigenin, and hesperetin) enantiomer contents in free form and bonded to glycosides by an RP‐UHPLC‐ESI‐MS/MS method. Different parts (stems, leaves, and flowers) of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.), and phacelia (Phacelia tanacetifolia Benth.) were used. The highest content of eriodictyol was found in goldenrod flowers (13.1 μg/g), where it occurred mainly as the (S)‐enantiomer, and the greatest proportion of the total amount was bonded to glycosides. The richest source of naringenin was found to be lucerne leaves (4.7 μg/g), where it was mainly bonded to glycosides and with the (S)‐enantiomer as the dominant form. Liquiritigenin was determined only in lucerne, where the flowers contained the highest amount (1.2 μg/g), with the (R)‐enantiomer as dominant aglycone form and the (S)‐enantiomer as the dominant glycosylated form. The highest hesperetin content was determined in phacelia leaves (0.38 μg/g), where it was present in the form of a glycoside and only as the (S)‐enantiomer. A comparison of the different analyte forms occurring in different plant parts was performed for the first time.