MS-based metabolite analysis of two licorice chalcones in mice plasma, bile, feces, and urine after oral administration.

Isoliquiritigenin (ILG) and isoliquiritin (ILQ), two kinds of major flavonoids in licorice, are biological active substances with antioxidant, anti-inflammatory, and tumor-suppressive effects. However, their in vivo metabolites, possible material basis of this two licorice chalcones for the treatment of diseases, have not been studied completely. To determine the metabolism of ILG and ILQ, after oral administration of 100 mg/kg/day of these compounds for consecutive 8 days, the metabolites of these two licorice chalcones in mice plasma, urine, feces, and bile were determined using liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry in this study. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism law, and reference standards-matching. As a result, a total of 25 and 29 metabolites of ILG and ILQ were identified, respectively. Seven main metabolic pathways, oxidation and reduction, deglycosylation and glycosylation, dehydroxylation and hydroxylation, demethoxylation and methoxylation, acetylation, glucuronidation, and sulfation, were summarized to tentatively explain how the metabolites were biologically transformed. These results provide the important information on the metabolism of ILG and ILQ, which may be helpful for the further research of their pharmacological mechanism.

Dalbergia ecastaphyllum (L.) Taub. and Symphonia globulifera L.f.: The Botanical Sources of Isoflavonoids and Benzophenones in Brazilian Red Propolis.

The Brazilian red propolis (BRP) constitutes an important commercial asset for northeast Brazilian beekeepers. The role of Dalbergia ecastaphyllum (L.) Taub. (Fabaceae) as the main botanical source of this propolis has been previously confirmed. However, in addition to isoflavonoids and other phenolics, which are present in the resin of D. ecastaphyllum, samples of BRP are reported to contain substantial amounts of polyprenylated benzophenones, whose botanical source was unknown. Therefore, field surveys, phytochemical and chromatographic analyses were undertaken to confirm the botanical sources of the red propolis produced in apiaries located in Canavieiras, Bahia, Brazil. The results confirmed D. ecastaphyllum as the botanical source of liquiritigenin (1), isoliquiritigenin (2), formononetin (3), vestitol (4), neovestitol (5), medicarpin (6), and 7-O-neovestitol (7), while Symphonia globulifera L.f. (Clusiaceae) is herein reported for the first time as the botanical source of polyprenylated benzophenones, mainly guttiferone E (8) and oblongifolin B (9), as well as the triterpenoids ß-amyrin (10) and glutinol (11). The chemotaxonomic and economic significance of the occurrence of polyprenylated benzophenones in red propolis is discussed.

Heterocycles 51: Liphophilicity investigation of some thiazole chalcones and aurones by experimental and theoretical methods.

Reversed-phase thin-layer chromatography and reversed-phase high-performance liquid chromatography were used for lipophilicity determination of a library of 30 thiazole chalcones and aurones previously synthetized in our laboratory. The experimental lipophilicity data have been compared with theoretical lipophilicity parameters estimated by various computational methods. Good correlations between the experimental and calculated lipophilicity parameters have been found for both investigated classes of compounds. Correlations between the lipophilicity of the thiazole chalcones and aurones and their antiproliferative activity were discussed. The methodologies and data gathered in this study will contribute to the lipophilicity studies of chalcones and aurones derivatives, two important classes of compounds in medicinal chemistry.

Model development for predicting in vitro bio-capacity of green rooibos extract based on composition for application as screening tool in quality control.

Mounting evidence of the ability of aspalathin to target underlying metabolic dysfunction relevant to the development or progression of obesity and type 2 diabetes created a market for green rooibos extract as a functional food ingredient. Aspalathin is the obvious choice as a chemical marker for extract standardisation and quality control, however, often the concentration of a single constituent of a complex mixture such as a plant extract is not directly related to its bio-capacity, i.e. the level of in vitro bioactivity effected in a cell system at a fixed concentration. Three solvents (hot water and two EtOH-water mixtures), previously shown to produce bioactive green rooibos extracts, were selected for extraction of different batches of rooibos plant material (n = 10). Bio-capacity of the extracts, tested at 10 µg ml-1, was evaluated in terms of glucose uptake by C2C12 and C3A cells and lipid accumulation in 3T3-L1 cells. The different solvents and inter-batch plant variation delivered extracts ranging in aspalathin content from 54.1 to 213.8 g kg-1. The extracts were further characterised in terms of other major flavonoids (n = 10) and an enolic phenylpyruvic acid glucoside, using HPLC-DAD. The 80% EtOH-water extracts, with the highest mean aspalathin content (170.9 g kg-1), had the highest mean bio-capacity in the respective assays. Despite this, no significant (P≥ 0.05) correlation existed between aspalathin content and bio-capacity, while the orientin, isoorientin and vitexin content correlated moderately (r≥ 0.487; P < 0.05) with increased glucose uptake by C2C12 cells. Various multivariate analysis methods were then applied with Evolution Program-Partial Least Squares (EP-PLS) resulting in models with the best predictive power. These EP-PLS models, based on all quantified compounds, predicted the bio-capacity of the extracts for the respective cell types with RMSECV values ≤ 11.5, confirming that a complement of compounds, and not aspalathin content alone, is needed to predict the in vitro bio-capacity of green rooibos extracts. Additionally, the composition of hot water infusions of different production batches of green rooibos (n = 29) at 'cup-of-tea' equivalence was determined to relate dietary supplementation with the extract to intake in the form of herbal tea.

Quantitation of dietary dihydrochalcones in Indian crabapple (Malus sikkimensis) using validated high-performance liquid chromatography.

In the present study, a systematic validated method was developed for the determination of two key dietary dihydrochalcones (DHC) viz. phloridzin (PZ) and phloretin (PT) in the leaves of Sikkim crabapple (Malus sikkimensis) using HPLC-Photo Diode Array (PDA). Chromatographic separation was optimized on a C18 column using a gradient elution of water/acetonitrile with the flow rate of 1.0 mL/min at 25°C at 280 nm. Sample preparation approach is rapid and energy efficient, and it requires no pre-concentration before analysis. Validation showed a good analytical performance in terms of specificity, linearity (r2 > 0.999), precision (% RSD < 1.08), recovery (97-100.4%) and sensitivities (limits of detection: 12.48 and 14.95 ng/mL; limit of quantification: 41.61 and 49.85 ng/mL) of PZ and PT, respectively. Developed approach was employed for targeted phytochemical analysis in the bark and fruits of M. sikkimensis. The PZ content in the bark and leaves was highest (12-13 mg/100 mg), about 90-fold higher than fruits. PT was only present in the leaves (0.57 mg/100 mg). The comparative data on PZ and PT content in various wild apple species/cultivar from different countries have also been discussed. The reliability of the validated method was established by analyzing global and expanded uncertainties in two DHC determinations in wild apple. The present method fulfills the technical requirement of ISO 17025:2017 for quality control of M. sikkimensis.

Licochalcone B Extracted from Glycyrrhiza uralensis Fisch Induces Apoptotic Effects in Human Hepatoma Cell HepG2.

The present study explored the molecular mechanism by which licochalcone B induces the cell cycle arrest and apoptosis in human hepatoma cell HepG2. Initial extraction and identification were performed by HPLC, UPLC-TOF-MS/MS, and NMR analysis, respectively. Licochalcone B inhibited the HepG2 growth with IC50 (110.15 µM) after 24 h, caused morphological distortion, and seized the cell cycle in the G2/M phase (cell arrest in G2/M:43.1 ± 2.2% for 120 µM versus 23.7 ± 1.2% for control), as well as induced apoptosis and intracellular ROS generation. Furthermore, exposure to licochalcone B markedly affected the cell cycle (up/down regulation) at mRNA and protein levels. Apoptosis was induced through the activation of receptor-mediated and mitochondrial pathways. The inhibition of Caspase 8 and Caspase 9 proteins abolished the licochalcone B induced apoptosis. The present work suggested that licochalcone B may further be identified as a potent functional food component with specific health benefits.

Visualization of Aspalathin in Rooibos (Aspalathus linearis) Plant and Herbal Tea Extracts Using Thin-Layer Chromatography.

Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts.

Isolation of flavonoids from apple peel using novel graphene oxide cotton fiber.

A novel graphene oxide cotton fibre (GOF) was used to adsorb flavonoids from crude ethanol extracts derived from apple peels. Ultra-high pressure liquid chromatography-mass spectrometry was used to analyse polyphenol content, and the resulting data demonstrated that GOF-based flash chromatography can be used to efficiently separate polyphenols from sugars and can facilitate the removal of 95% of the sugar content. Flavonoids can be easily separated from phenolic acids. Chalcones and flavonols were eluted with 100% methanol and subsequently flavan-3-ols can be eluted with 0.04 M sodium hydroxide. The novel GOF has the potential to be used in the isolation of flavonoids.

Industrial processing versus home processing of tomato sauce: Effects on phenolics, flavonoids and in vitro bioaccessibility of antioxidants.

The effect of industrial and home processing, in vitro gastrointestinal digestion, individual phenolic content, and antioxidant capacity of tomato into tomato sauce were investigated. Industrial processing of tomato fruit into sauce had an overall positive effect on the total antioxidant capacity (∼1.2-fold higher) compared to tomato fruit whereas home processing of tomato fruit into sauce led to a decrease in these values. Untargeted LC-QTOF-MS analysis revealed 31 compounds in tomato that changed upon processing, of which 18 could be putatively identified. Naringenin chalcone is only detectable in the fruit, while naringenin is strongly increased in the sauces. Rutin content increased by 36% in the industrial processed sauce whereas decreased by 26% in the home processed sauce when compared to fruit. According to the results of an in vitro gastrointestinal digestion model, industrial processing may lead to enhanced bioaccessibility of antioxidants.

Rapid Identification and Comparison of Compounds with Antioxidant Activity in Coreopsis tinctoria Herbal Tea by High-Performance Thin-Layer Chromatography Coupled with DPPH Bioautography and Densitometry.

A simple and efficient method based on high-performance thin-layer chromatography coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioautography (HPTLC-DPPH) was established for the screening and comparison of antioxidants in different parts of Coreopsis tinctoria herbal tea from different origins and other related herbal tea materials, which used Chrysanthemum morifolium cv. "Gongju" and "Hangju" in this study. Scanning densitometry after DPPH derivatization was applied for the determination of antioxidant capacities of isolated compounds in each sample. It is considered that ethanol extracts of C. tinctoria had stronger antioxidant activity and more characteristic bands than those of 2 compared samples, C. morifolium cv. "Gongju" and "Hangju." Chemometric analysis results showed that the combination of hierarchical clustering analysis and principal component analysis based on determined antioxidant capacities could be used for the discrimination of different parts of C. tinctoria and C. morifolium. Results showed that 7 compounds made up the major contributions of antioxidant activity in C. tinctoria, including okanin, isookanin, marein, flavanomarein, 5,7,3',5'-tetrahydroxyflavanone-7-O-glucoside, 3,5-dicaffeoylquinic acid, and chlorogenic acid. Therefore, 7 compounds were identified as major antioxidant biomarkers for quality control of C. tinctoria. Results demonstrated that the established method could be applied for the identification of C. tinctoria, and were beneficial for the bioactivity-based quality control of C. tinctoria.

[Determination of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Quantitative analysis of flavanones and chalcones from willow bark.

Willow bark extracts are used for the treatment of fever, pain and inflammation. Recent clinical and pharmacological research revealed that not only the salicylic alcohol derivatives, but also the polyphenols significantly contribute to these effects. Quantitative analysis of the European Pharmacopoeia still focuses on the determination of the salicylic alcohol derivatives. The objective of the present study was the development of an effective quantification method for the determination of as many flavanone and chalcone glycosides as possible in Salix purpurea and other Salix species as well as commercial preparations thereof. As Salix species contain a diverse spectrum of the glycosidated flavanones naringenin, eriodictyol, and the chalcone chalconaringenin, a subsequent acidic and enzymatic hydrolysis was developed to yield naringenin and eriodictyol as aglycones, which were quantified by HPLC. The 5-O-glucosides were cleaved with 11.5% TFA before subsequent hydrolysis of the 7-O-glucosides with an almond ß-glucosidase at pH 6-7. The method was validated with regard to LOD, LOQ, intraday and interday precision, accuracy, stability, recovery, time of hydrolysis, robustness and applicability to extracts. All 5-O- and 7-O-glucosides of naringenin, eriodictyol and chalconaringenin were completely hydrolysed and converted to naringenin and eriodictyol. The LOD of the HPLC method was 0.77 µM of naringenin and 0.45 µM of eriodictyol. The LOQ was 2.34 µM of naringenin and 1.35 µM for eriodictyol. The method is robust with regard to sample weight, but susceptible concerning enzyme deterioration. The developed method is applicable to the determination of flavanone and chalcone glycosides in willow bark and corresponding preparations.

Biological activities of polyphenols from Polygonum lapathifolium / Actividades biológicas de polifenoles de Polygonum lapathifolium

Polygonum lapathifolium is an invasive plant spread worldwide, which has been used in traditional medicine for its biological activities. We studied chemical profile of the aerial part using HPLC/DAD/ESI-MS. Eight flavonoids, two chalcones and gallic acid were isolated, identified nad tested for their activity in seven experimental models (for antioxidant, alpha/beta-glucosidase inhibitory, antimicrobial, anticholineserase activity). Our results showed that P. lapathifolium is a valuable source of compounds with perspective biological activities...

Polygonum lapathifolium es una planta invasora cuya presencia se extiende por todo el mundo y que ha sido utilizada en la medicina tradicional por sus actividades biológicas. Analizamos el perfil químico de la parte superficial utilizando HPLC/DAD/ESI-MS. Ocho flavonoides, dos chalconas y ácido gálico fueron aislados, identificados y analizados en relación a sus actividades en siete modelos experimentales (de actividad antioxidante, alfa/beta-glucosidasa inhibitoria, antimicrobiana, anticholineserasa). Nuestros resultados demuestran que P. lapathifolium es una valiosa fuente de compuestos con actividades biológicas prometedoras...

Qualitative identification of dibenzoylmethane in licorice root (Glycyrrhiza glabra) using gas chromatography-triple quadrupole mass spectrometry.

Licorice root (Glycyrrhiza glabra), an herbal Chinese medicine, has shown medicinal uses in therapeutics and cancer prevention. Dibenzoylmethane (DBM; 1, 3-diphenyl-1, 3-propadinedione), a small beta-diketone, has been reported to be a minor constituent of licorice and a known deregulator of the human prostate cancer cell cycle. Characterization of the phytochemical profiles of licorice root forms including commercially available DBM will advance our search in identifying novel reagents for prostate cancer therapeutics. Gas chromatography- triple quadrupole-mass spectrometric analysis was used for detecting DBM in licorice root extracts. DBM and all licorice forms exhibited a component with a retention time of 14.5 minutes. The major fragment ions detected were at m/z 77, 105, 147, 223 and 224 at the identified retention time by selected reaction monitoring/SRM. These data confirm the presence of DBM from its natural source (G. glabra), and the GC-MS/SRM method helps in the identification of this minor component in a complex biological matrix.

Special licorice extracts containing lowered glycyrrhizin and enhanced licochalcone A prevented Helicobacter pylori-initiated, salt diet-promoted gastric tumorigenesis.

OBJECTIVE: In spite of cytoprotective and anti-inflammatory actions, conventional licorice extracts (c-lico) were limitedly used due to serious side effects of glycyrrhizin. As our group had successfully isolated special licorice extracts (s-lico) lowering troublesome glycyrrhizin, but increasing licochalcone A, we have compared anti-inflammatory, antioxidative, and cytoprotective actions of s-lico and c-lico against either in vitro or in vivo Helicobacter pylori infection. METHODS: RT-PCR and Western blot were performed to check anti-inflammatory action and electron spin resonance (ESR) and DCFDA spectroscopy to check antioxidative action. s-lico or c-lico was pretreated 1 hours before H. pylori infection on AGS cells. Interleukin-10 deficient mice inoculated H. pylori and followed with high salt containing pallet diets to produce H. pylori-associated chronic atrophic gastritis and gastric tumors, during which s-lico or c-lico-containing pellet diets were administered up to 24 weeks. RESULTS: s-lico had fabulous efficacy on scavenging ROS which was further confirmed by DCFDA study and ESR measurement. The expressions of COX-2, iNOS, VEGF, and IL-8 were increased after H. pylori infection, of which levels were significantly decreased with s-lico in a dose-dependent manner. s-lico significantly ameliorated hypoxia-induced or H. pylori-induced angiogenic activities. s-lico significantly ameliorated H. pylori-induced gastric damages as well as gastritis. Our animal model showed significant development of gastric tumors including adenoma and dysplasia relevant to H. pylori infection, and s-lico administration significantly attenuated incidence of H. pylori-induced gastric tumorigenesis. CONCLUSIONS: Special licorice extracts can be anticipating substance afforded significant attenuation of either H. pylori-induced gastritis or tumorigenesis based on potent antioxidative, anti-inflammatory, and antimutagenic actions.

Home processing of tomatoes (Solanum lycopersicum): effects on in vitro bioaccessibility of total lycopene, phenolics, flavonoids, and antioxidant capacity.

BACKGROUND: In order to investigate the effect of home processing on the bioaccessibility of health-related constituents of tomatoes, total lycopene, phenolics, flavonoids and antioxidant capacity were determined from seven different tomato products using an in vitro gastrointestinal digestion model. Additionally, the changes in the contents of the major tomato phenolics were determined and compared for these different tomato products using HPLC. RESULTS: The results revealed that paste processing and drying significantly increased the bioaccessible total lycopene content (2.2- and 3.8-fold, respectively), total phenolic content (2.3- and 2.0-fold, respectively), total flavonoid content (9.0- and 2.5-fold, respectively) and total antioxidant capacity (6.3- and 8.0-fold for the DPPH assay, 26- and 33-fold for the CUPRAC assay, respectively) (P < 0.05) compared to fresh tomatoes. HPLC analysis revealed significantly lower (P < 0.05) rutin content in puree and juice. The loss of naringenin chalcone in some tomato products, as well as its conversion into naringenin in heat-treated products was observed. CONCLUSION: The current study provided valuable insights into the changes in the content and bioaccessibility of tomato antioxidants as a result of home processing.

Synthesis and cytotoxicity of chalcones and 5-deoxyflavonoids.

Chalcones 1~8 and 5-deoxyflavonoids 9~22 were synthesized in good yields by aldol condensation, Algar-Flynn-Oyamada reaction, glycosidation, and deacetylation reaction, respectively, starting from 2-acetyl phenols substituted by methoxy or methoxymethoxy group and appropriately benzaldehydes substituted by methoxy, methoxymethoxy group, or chlorine. Among them, 13 and 17~22 are new compounds. The cytotoxicity bioassays of these chalcones and 5-deoxyflavonoids were screened using the sulforhodamine B (SRB) protein staining method, and the results showed that compounds 2, 4, 5, 6, 10, 15, and 19 exhibited moderate cytotoxicity against the cancer cell line of MDA-MB-231, U251, BGC-823, and B16 in comparison with control drugs (HCPT, Vincristine, and Taxol).

Electrospray tandem mass spectrometry analysis of methylenedioxy chalcones, flavanones and flavones.

RATIONALE: Several methylenedioxy chalcones, flavanones and flavones substituted with mono-, di- and trimethoxy groups have been used in the treatment of proliferative conditions like cancer and inflammatory diseases. The application of these flavonoids in biology requires an analytical method to ensure a detailed knowledge of their structures after drug metabolism. METHODS: Electrospray ionization mass (ESI-MS) and tandem mass (ESI-MS/MS) spectra were acquired using a Q-TOF 2 instrument. Fragmentation patterns and their pathways were analyzed by CID-MS(2-3) spectra acquired in a LXQ linear ion trap mass spectrometer using standard isolation and excitation procedures (activation q value of 0.25, activation time of 30 ms). ESI-MS and ESI-MS(n) conditions: spray voltage 5 kV, nitrogen 8.00 sheath gas flow rate (arb), heated capillary temperature 275°C, capillary voltage 10.99 V; tube lens voltage 75.01 V. RESULTS: The ESI-MS/MS spectra of chalcones were nearly identical to their corresponding isomeric flavanones with (0,α)A(+)/(1,3)A(+) and (0,1')B(+)/(1,4)B(+) cleavages. Other common losses are of (•)CH3, H2O, HCHO and C2H2O. The characteristic loss of C2H2O and absence of a (0,α)B(+)/(1,3)B(+) product ion allows to distinguish between the 2- or 4-methoxy-substituted chalcones and flavanones. Common losses of (•)CH3, (•)CH3 and (•)H, and C2H2O2 characteristic for the presence of methylenedioxy groups were observed in flavones. CONCLUSIONS: The substitution pattern on the B-ring leads to distinct base peak formation in the flavones. In addition, differentiation of isomers with methoxy substituents in ortho and para positions of the B-ring was achieved using MS/MS in chalcones and flavanones. This method will be helpful for identification of these compounds in biological mixtures.

Dynamic residual complexity of the isoliquiritigenin-liquiritigenin interconversion during bioassay.

Bioactive components in food plants can undergo dynamic processes that involve multiple chemical species. For example, 2'-hydroxychalcones can readily isomerize into flavanones. Although chemically well documented, this reaction has barely been explored in the context of cell-based assays. The present time-resolved study fills this gap by investigating the isomerization of isoliquiritigenin (a 2'-hydroxychalcone) and liquiritigenin (a flavanone) in two culture media (Dulbecco's modified eagle medium and Roswell Park Memorial Institute medium) with and without MCF-7 cells, using high-performance liquid chromatography-diode array detector-electrospray ionization/atmospheric pressure chemical ionization-mass spectrometry for analysis. Both compounds were isomerized and epimerized under all investigated biological conditions, leading to mixtures of isoliquiritigenin and R/S-liquiritigenin, with 19.6% R enantiomeric excess. Consequently, all three species can potentially modulate the biological responses. This exemplifies dynamic residual complexity and demonstrates how both nonchiral reactions and enantiomeric discrimination can occur in bioassay media, with or without cells. The findings highlight the importance of controlling in situ chemical reactivity, influenced by biological systems when evaluating the mode of action of bioactives.

Comprehensive two-dimensional gas chromatography, retention indices and time-of-flight mass spectra of flavonoids and chalcones.

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC×GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC×GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 µg/mL. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC×GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum.