Exploring the Chemistry of Ocimum Species under Specific Extractions and Chromatographic Methods: A Systematic Review.

Ocimum is considered the largest genus in the Lamiacea family. The genus includes basil, a group of aromatic plants with a wide range of culinary uses that nowadays draws attention for its medicinal and pharmaceutical potential. This systematic review intends to explore the chemical composition of nonessential oils and their variation across different Ocimum species. Moreover, we aimed to identify the state of knowledge regarding the molecular space in this genus as well as the different methods of extraction/identification and geographical location. Seventy-nine eligible articles were selected for the final analysis, from which we extracted more than 300 molecules. We found that the countries with the highest number of studies into Ocimum species are India, Nigeria, Brazil, and Egypt. However, from all known species of Ocimum, only 12 were found to have an extensive chemical characterization, particularly Ocimum basilicum and Ocimum tenuiflorum. Our study focused especially on alcoholic, hydroalcoholic, and water extracts, in which the main techniques for compound identifications are GC-MS, LC-MS, and LC-UV. Across the compiled molecules, we found a wide variety of compounds, especially flavonoids, phenolic acids, and terpenoids, suggesting that this genus could be a very useful source of possible bioactive compounds. The information collected in this review also emphasizes the huge gap between the vast number of Ocimum species discovered and the number of studies in each of them that determined the chemical characterization.

Characterization of polyphenols in the methanol leaf extract of Bathysa gymnocarpa (Rubiaceae) by HPLC-DAD-ESI-MS/MS.

The species Bathysa gymnocarpa K.Schum is a tree belonging to the Rubiaceae family, endemic in Brazil. So far, there are reports neither of phytochemical work nor of biological evaluation of it. The analysis by High Performance Liquid Chromatography coupled to a Diode Array Detector and a tandem Mass Spectrometer with an Electrospray Ionization source (HPLC-DAD-ESI-MS/MS) of its crude extract allowed to characterize in a complex mixture, without isolation, fourteen compounds, being two as cinnamic acid derivatives, and the others as mono-, di- and triglycosilated derivatives of the flavonols quercetin and kaempferol. These compounds are reported for the first time in Bathysa spp.

A Liposomal Formulation to Exploit the Bioactive Potential of an Extract from Graciano Grape Pomace.

Antioxidant compounds with health benefits can be found in food processing residues, such as grape pomace. In this study, antioxidants were identified and quantified in an extract obtained from Graciano red grape pomace via a green process. The antioxidant activity of the extract was assessed by the DPPH and FRAP tests, and the phenolic content by the Folin-Ciocalteu test. Furthermore, nanotechnologies were employed to produce a safe and effective formulation that would exploit the antioxidant potential of the extract for skin applications. Anthocyanins, flavan-3-ols and flavanols were the main constituents of the grape pomace extract. Phospholipid vesicles, namely liposomes, were prepared and characterized. Cryo-TEM images showed that the extract-loaded liposomes were predominantly spherical/elongated, small, unilamellar vesicles. Light scattering results revealed that the liposomes were small (~100 nm), homogeneously dispersed, and stable during storage. The non-toxicity of the liposomal formulation was demonstrated in vitro in skin cells, suggesting its possible safe use. These findings indicate that an extract with antioxidant properties can be obtained from food processing residues, and a liposomal formulation can be developed to exploit its bioactive value, resulting in a promising healthy product.

Untargeted Metabolomic Liquid Chromatography High-Resolution Mass Spectrometry Fingerprinting of Apple Cultivars for the Identification of Biomarkers Related to Resistance to Rosy Apple Aphid.

Liquid chromatography high-resolution mass spectrometry fingerprinting together with pattern recognition techniques was used to determine the metabolites involved in the susceptibility of apple cultivars to rosy apple aphid (RAA). Preprocessing of ultra-high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry raw data of resistant and susceptible apple cultivars was carried out with XCMS and CAMERA packages. Univariate statistical tools and multivariate data analysis highlighted significant different profiles of the apple metabolomes according to their tolerance to RAA. Optimized and cross-validated Partial least squares discriminant analysis and orthogonal projections to latent structures discriminant analysis models confirmed trans-4-caffeoylquinic acid and 4-p-coumaroylquinic acid as biomarkers for the identification of resistant and susceptible apple cultivars to RAA and disclosed that only hydroxycinnamic acids are involved in the disease susceptibility of cultivars. In this sense, the final steps of the biosynthesis of caffeoylquinic acid (CQA) and p-coumaroylquinic acid (p-CoQA) become decisive because the isomerization of 5-CQA to 4-CQA is favored in resistant cultivars, whereas the isomerization of 5-p-CoQA to 4-p-CoQA is favored in susceptible cultivars.

Metabolomic profile and computational analysis for the identification of the potential anti-inflammatory mechanisms of action of the traditional medicinal plants Ocimum basilicum and Ocimum tenuiflorum.

Ocimum basilicum and Ocimum tenuiflorum are two basil species widely used medicinally as an anti-inflammatory, antimicrobial and cardioprotective agent. This study focuses on the chemical characterization of the majoritarian compounds of both species and their anti-inflammatory potential. Up to 22 compounds such as various types of salvianolic acids, derivatives of rosmaniric acid and flavones were identified in both plants. The identified compounds were very similar between both plants and are consistent with previous finding in other studies in Portugal and Italy. Based on the identified molecules a consensus target prediction was carried out. Among the main predicted target proteins, we found a high representation of the carbonic anhydrase family (CA2, CA7 and CA12) and several key proteins from the arachidonic pathway (LOX5, PLA2, COX1 and COX2). Both pathways are well related to inflammation. The interaction between the compounds and these targets were explored through molecular docking and molecular dynamics simulation. Our results suggest that some molecules present in both plants can induce an anti-inflammatory response through a non-steroidal mechanism of action connected to the carbon dioxide metabolism.

Tempranillo Grape Extract in Transfersomes: A Nanoproduct with Antioxidant Activity.

Polyphenols are gaining increasing interest due to their beneficial properties to human health. Grape pomace, the by-product of wine production, is a source of these bioactive compounds. An extract from Tempranillo grape pomace was obtained and characterized qualitatively and quantitatively. The major components found were anthocyanins, flavan-3-ols, and flavonols. To improve the bioavailability of these compounds, the extract was formulated in phospholipid vesicles, namely transfersomes. Spherical unilamellar vesicles around 100 nm each were obtained. The antioxidant activity of both the extract and the transfersomes was evaluated by using colorimetric assays (i.e., DPPH, FRAP, and Folin-Ciocalteu). The cells' viability and the antioxidant activity were assessed in keratinocytes. The results showed that the extract and the transfersomes had no cytotoxic effects and exerted remarkable antioxidant activity, which was more evident in a vesicle formulation. These findings highlighted the potential of the Tempranillo grape pomace extract and the efficacy of the incorporation into phospholipid vesicles.

Comprehensive characterisation of polyphenols in leaves and stems of three anti-dengue virus type-2 active Brazilian Faramea species (Rubiaceae) by HPLC-DAD-ESI-MS/MS.

INTRODUCTION: The methanol (MeOH) leaf extracts of the species Faramea bahiensis, F. hyacinthina and F. truncata (Rubiaceae) have previously shown in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activities in human hepatocarcinoma cell lineage (HepG2). Chemical studies have led to the isolation of major flavonoids, but quite complex fractions of phenolic compounds still remain. OBJECTIVE: To complete the study of phenolic compounds in the leaves and to access the presence of these compounds in the stems of these Faramea spp. by online high-performance liquid chromatography-diode array detector-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS), as well as to evaluate the in vitro cytotoxic and anti-DENV2 activities of their MeOH stem extracts. METHODOLOGY: The identification was performed by comparing retention times, UV and mass spectra with those of available standards and by using the mechanisms and fragmentation patterns established in previous studies. The effects of the extracts in DENV2 infected HepG2 cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The virus titer was quantified by plaque assay. RESULTS: The study led to the characterisation of 31 phenolic compounds including flavonoid O- and C-glycosides, phenolic acids and one coumarin. The stem extracts from F. hyacinthina and F. bahiensis presented a similar bioactivity to those of their leaves but a loss of cytoprotective activity of F. bahiensis and a higher cytotoxicity of F. truncata were observed. CONCLUSIONS: This research allowed a detailed phenolic composition of three bioactive Faramea species to be achieved, thus contributing to the study of this genus and providing valuable information for further phytotherapeutic applications.

Relationship between hydroxycinnamic acids and the resistance of apple cultivars to rosy apple aphid.

The phenolic profiles of apple cultivars from the SERIDA Asturian cider apple breeding program, including parents and progenies, were determined by ultrahigh-performance liquid chromatography-diode array detector-electrospray ionization-quadrupole time of flight/mass spectrometer in order to study the relationship between phenols and the resistance of apple tree cultivars to rosy apple aphid (RAA). A pattern recognition technique named partial least square discriminant analysis (PLS-DA) was used to classify apple cultivars based on resistance to RAA, resistant and susceptible, reaching scores with accuracy higher than 97% and 91% respectively. Hydroxycinnamic acids, particularly 4-caffeoylquinic acid (4-CQA) and 4-p-coumaroylquinic acid (4-pCoQA), were identified as the major player in RAA resistance by the PLS-DA model. Indeed, the isomerisation 5-CQA → 4-CQA is favoured in resistant cultivars, whereas the isomerisation 5-pCoQA → 4-pCoQA is favoured in susceptible cultivars. As a result, resistant cultivars accumulate higher amounts of 4-CQA than susceptible ones, and the opposite occurs for 4-pCoQA. Also, minor isomerisations of 5-CQA to 1-CQA or 3-CQA show opposite behaviour for resistant and susceptible cultivars. Cultivar resistance to RAA is concluded to be related with the phenylpropanoid pathway, the isomerisation reactions being the key metabolic reaction for a cultivar to be resistant or susceptible to RAA.

Polyphenolic profile of butterhead lettuce cultivar by ultrahigh performance liquid chromatography coupled online to UV-visible spectrophotometry and quadrupole time-of-flight mass spectrometry.

In the present study, the butterhead lettuce cultivar was analyzed by ultrahigh performance liquid chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry (QToF/MS) in the positive and negative ion mode in order to characterize its polyphenolic profile for the first time. The instrument acquisition mode MSE was used to collect automatic and simultaneous information of exact mass at high and low collision energies of precursor ions as well as other ions produced as a result of their fragmentation. One hundred eleven phenolic compounds were identified in the acidified hydromethanolic extract of freeze-dried leaves of butterhead lettuce cultivar: 40 hydroxycinnamic acid derivatives, 21 hydroxybenzoic acid derivatives, 2 hydroxyphenylacetic acid derivatives, 18 flavonols, 9 flavones, one flavanone, 7 coumarins, one hydrolysable tannin and 12 lignans. Forty-seven of these compounds have been tentatively identified for the first time in lettuce.

Characterization of phenolic compounds in green and red oak-leaf lettuce cultivars by UHPLC-DAD-ESI-QToF/MS using MSE scan mode.

Lettuce (Lactuca sativa) is one of the most popular leafy vegetables in the world and constitutes a major dietary source of phenolic compounds with health-promoting properties. In particular, the demand for green and red oak-leaf lettuces has considerably increased in the last years but few data on their polyphenol composition are available. Moreover, the usage of analytical edge technology can provide new structural information and allow the identification of unknown polyphenols. In the present study, the phenolic profiles of green and red oak-leaf lettuce cultivars were exhaustively characterized by ultrahigh-performance liquid chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization (ESI), and quadrupole time-of-flight mass spectrometry (QToF/MS), using the MSE instrument acquisition mode for recording simultaneously exact masses of precursor and fragment ions. One hundred fifteen phenolic compounds were identified in the acidified hydromethanolic extract of freeze-dried lettuce leaves. Forty-eight of these compounds were tentatively identified for the first time in lettuce, and only 20 of them have been previously reported in oak-leaf lettuce cultivars in literature. Both oak-leaf lettuce cultivars presented similar phenolic composition, except for apigenin-glucuronide and dihydroxybenzoic acid, only detected in the green cultivar; and for luteolin-hydroxymalonylhexoside, an apigenin conjugate with molecular formula C40 H54 O19 (monoisotopic MW = 838.3259 u), cyanidin-3-O-glucoside, cyanidin-3-O-(3″-O-malonyl)glucoside, cyanidin-3-O-(6″-O-malonyl)glucoside, and cyanidin-3-O-(6″-O-acetyl)glucoside, only found in the red cultivar. The UHPLC-DAD-ESI-QToF/MSE approach demonstrated to be a useful tool for the characterization of phenolic compounds in complex plant matrices.

On line characterization of 58 phenolic compounds in Citrus fruit juices from Spanish cultivars by high-performance liquid chromatography with photodiode-array detection coupled to electrospray ionization triple quadrupole mass spectrometry.

Polyphenol profile of Citrus juices of sweet orange, tangerine, lemon and grapefruit from Spanish cultivars was obtained by High-Performance Liquid Chromatography with Diode Array Detection coupled to Electrospray ionization and Triple Quadrupole Mass Spectrometry. Fifty eight phenolic compounds of five different classes were identified in these Citrus juices. Flavanone: O-dihexoside of naringenin; flavones: apigenin-7-O-rutinoside-4'-O-glucoside, luteolin-7-O-neohesperidoside-4'-O-glucoside, luteolin-6-C-glucoside, 6,8-di-C-acylhexosides of chrysoeriol and diosmetin, 6C- and 8C-glucoside-O-pentoside of apigenin, apigenin-6-C-hexoside-O-hexoside and apigenin-8-C-hexoside-O-acylrhamnoside; flavonols: 7-O-rutinosides of quercetin, kaempferol, isorhamnetin and tamarixetin, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside-7-O-glucoside, tamarixetin-3-O-rutinoside-7-O-glucoside, isorhamnetin-3-O-hexoside-7-O-rhamnosylhexoside, 3-O-rhamnoside-7-O-rhamnosylhexoside of quercetin and isorhamnetin and kaempferol-3-O-rhamnosylhexoside-7-O-rhamnoside; hydroxycinnamic acids: O-hexoside of ferulic and sinapic acid; and, coumarins: O-hexoside and O-rhamnosylhexoside of scopoletin, had not previously been reported in Citrus juices to our knowledge. Structures have been assigned on the basis of the complementary information obtained from retention time, UV-visible spectra, scan mode MS spectra, and fragmentation patterns in MS(2) spectra obtained using different collision energies. A structure diagnosis scheme is provided for the identification of different phenolic compounds.

Chemometric characterization of fruit juices from Spanish cultivars according to their phenolic compound contents: I. Citrus fruits.

The data set composed by phenolic compound profiles of 83 Citrus juices (determined by HPLC-DAD-MS/MS) was evaluated by chemometrics to differentiate them according to Citrus species (sweet orange, tangerine, lemon, and grapefruit). Cluster analysis (CA) and principal component analysis (PCA) showed natural sample grouping among Citrus species and even the Citrus subclass. Most of the information contained in the full data set can be captured if only 15 phenolic compounds (concentration ≥10 mg/L), which can be quantified with fast and accurate methods in real samples, are introduced in the models; a good classification which allows the confirmation of the authenticity of juices is achieved by linear discriminant analysis. Using this reduced data set, fast and routine methods have been developed for predicting the percentage of grapefruit in adulterated sweet orange juices using principal component regression (PCR) and partial least-squares regression (PLS). The PLS model has provided suitable estimation errors.

A fragmentation study of dihydroquercetin using triple quadrupole mass spectrometry and its application for identification of dihydroflavonols in Citrus juices.

A mass spectrometric method using electrospray ionization with triple quadrupole and quadrupole time-of-flight hybrid (Q-Tof) mass spectrometry has been applied to the structural characterization of dihydroflavonols. This family of compounds has been studied by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the first time in this work. A comprehensive study of the product ion MS spectra of the [M+H](+) ion of a commercially available standard has been performed. The most useful fragmentations in terms of structural identification are those that involve cleavage of the C-ring, resulting in diagnostic ions of dihydroflavonol family: (1,3)A(0) (+), (1,2)B(0) (+), (1,2)B(0) (+)-CO, (0,2)A(0) (+), (0,2)A(0) (+)-H(2)O, (0,2)A(0) (+)-CO, and (0,2)A(0) (+)-H(2)O-CO, that allow the characterization of the substituents in the A- and B-rings. In addition to those ions, other product ions due to losses of H(2)O and CO molecules from the Y(0) (+) ion were observed. Their fragmentation mechanisms and ion structures have been proposed. The established fragmentation patterns have been used to successfully identity three dihydroflavonols found in tangerine juices for the first time.

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry.

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV-visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y(0)](+). This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.

Liquid chromatography coupled with ultraviolet absorbance detection, electrospray ionization, collision-induced dissociation and tandem mass spectrometry on a triple quadrupole for the on-line characterization of polyphenols and methylxanthines in green coffee beans.

Liquid chromatography coupled with a photodiode array detector, electrospray ionization, collision-induced dissociation and tandem mass spectrometry (LC-DAD/ESI-CID-MS/MS) on a triple quadrupole (QqQ) has been used to detect and characterize polyphenols and methylxanthines in green coffee beans: three phenolic acids (caffeic acid, ferulic acid and dimethoxycinnamic acid), three isomeric caffeoylquinic acids (M(r) 354), three feruloylquinic acids (M(r) 368), one p-coumaroylquinic acid (M(r) 338), three dicaffeoylquinic acids (M(r) 516), three feruloyl-caffeoylquinic acids (M(r) 530), four p-coumaroyl-caffeoylquinic acids (M(r) 500), three diferuloylquinic acids (M(r) 544), six dimethoxycinnamoyl-caffeoylquinic acids (M(r) 544), three dimethoxycinnamoyl-feruloylquinic acids (M(r) 558), six cinnamoyl-amino acid conjugates, three cinnamoyl glycosides, and three methylxanthines (caffeine, theobromine and theophylline). Dimethoxycinnamic acid, three isomers of dimethoxycinnamoyl-caffeoylquinic acids and another three of dimethoxycinnamoyl-feruloylquinic acids, as well as the three cinnamoyl glycosides, had not previously been reported in coffee beans. Structures have been assigned on the basis of the complementary information obtained from UV-visible spectra, relative hydrophobicity, scan mode MS spectra, and fragmentation patterns in MS(2) spectra (both in the positive and negative ion modes) obtained using a QqQ at different collision energies. A structure diagnosis scheme is provided for the identification of different isomers of polyphenols and methylxanthines.

New features on the fragmentation and differentiation of C-glycosidic flavone isomers by positive electrospray ionization and triple quadrupole mass spectrometry.

Six flavone mono-C-glucosides, four standards (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)- apigenin and luteolin) and two others from lemon juice (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)-diosmetin) have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high-performance liquid chromatography with diode-array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Several systematic differences between collision-induced dissociation tandem mass (CID-MS/MS) spectra of C-6- and C-8-isomers have been found and some general guidelines and two new diagnostic product ions have been proposed for the differentiation of C-6- and C-8-flavonoid glycosides. These results have been successfully applied to the characterization of two flavone C-glycosides found in lemon juice, and mass spectra of a flavone di-C-glycoside detected in lemon juice have been studied and interpreted.

Supervised pattern recognition in food analysis.

Data analysis has become a fundamental task in analytical chemistry due to the great quantity of analytical information provided by modern analytical instruments. Supervised pattern recognition aims to establish a classification model based on experimental data in order to assign unknown samples to a previously defined sample class based on its pattern of measured features. The basis of the supervised pattern recognition techniques mostly used in food analysis are reviewed, making special emphasis on the practical requirements of the measured data and discussing common misconceptions and errors that might arise. Applications of supervised pattern recognition in the field of food chemistry appearing in bibliography in the last two years are also reviewed.

A validated solid-liquid extraction method for the HPLC determination of polyphenols in apple tissues Comparison with pressurised liquid extraction.

A solid-liquid extraction procedure followed by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector (DAD) for the determination of polyphenols in freeze-dried apple peel and pulp is reported. The extraction step consists in sonicating 0.5g of freeze-dried apple tissue with 30mL of methanol-water-acetic acid (30:69:1, v/v/v) containing 2g of ascorbic acid/L, for 10min in an ultrasonic bath. The whole method was validated, concluding that it is a robust method that presents high extraction efficiencies (peel: >91%, pulp: >95%) and appropriate precisions (within day: R.S.D. (n = 5) <5%, and between days: R.S.D. (n = 5) <7%) at the different concentration levels of polyphenols that can be found in apple samples. The method was compared with one previously published, consisting in a pressurized liquid extraction (PLE) followed by RP-HPLC-DAD determination. The advantages and disadvantages of both methods are discussed.

Technological classification of basque cider apple cultivars according to their polyphenolic profiles by pattern recognition analysis.

The polyphenolic compositions of 31 Basque cider apple cultivars were determined in pulp, peel, and juice by high-performance liquid chromatography--diode array detection analysis of crude extracts and after thiolysis. Data sets, consisting of individual polyphenol concentrations, total procyanidin content, and the average degree of polymerization of procyanidins, were evaluated by multivariate chemometric techniques, to develop decision rules for classifying apple cultivars technologically into bitter and nonbitter categories. A preliminary study of the data structure was performed by cluster analysis and principal component analysis in each apple material. Bitter apple varieties presented higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars. Different classification systems for the two categories on the basis of the chemical data were obtained applying several supervised pattern recognition procedures, such as linear discriminant analysis, K-nearest neighbors, soft independent modeling of class analogy, partial least-squares, and multilayer feed forward artificial neural networks. Excellent performance in terms of recognition and prediction abilities for both categories (100% of hits) was achieved in every case (pulp, peel, or juice). Polyphenolic profiles of apple pulp, peel, or juice provide enough information to develop classification criteria for establishing the technological group of apple cultivars (bitter or nonbitter).

On-line characterisation of apple polyphenols by liquid chromatography coupled with mass spectrometry and ultraviolet absorbance detection.

Apple polyphenols were characterised by means of hyphenated techniques such as HPLC coupled to UV photodiode array detection (LC-DAD) and to mass spectrometry (LC-MS). LC-MS using atmospheric pressure ionisation (APCI) in the positive ion mode provided the molecular weight, the number of hydroxyl groups, the number of sugars and an idea about the substitution pattern of the polyphenols. LC-DAD with postcolumn addition of UV shift reagents afforded precise structural information about the position of the free hydroxyl groups in the polyphenolic nucleus. Five isorhamnetin glycosides, two hydroxyphloretin glycosides and quercetin were reported in apple peel for the first time. Postcolumn addition of UV shift reagents in LC-DAD analysis confirmed the presence of isorhamnetin glycosides and not the isomeric glycosides of rhamnetin. Moreover, isorhamnetin-3-O-rhamnoglucoside was identified unambiguously by comparison with a standard. These results are relevant not only from a chemotaxonomic point of view, but also in the control of authenticity of fruit derived products in order to detect fraudulent admixtures.