HPLC separation of flavonols, flavones and oxidized flavonols with UV-, DAD-, electrochemical and ESI-ion trap MS detection

The cation-induced or electrochemical oxidation of flavonols has been reported to yield 2-(hydroxybenzoyl)-2-hydroxy-3(2H)-benzofuranones. Two new gradient reversed phase HPLC methods are presented which allow the determination of those oxidized flavonols simultaneously with flavonols and flavones. UV and electrochemical detection are used because of their high sensitivity. Qualitative detection together with quantification of all compounds is achieved with photodiode-array detection. An electrospray ionization ion trap mass spectrometric method is presented for unique identification of the benzofuranones after HPLC separation.     

Bioaccumulation and biotransformation of flavonols by erythrocytes

A model for the complete system of bioaccumulation, transport, and biotransformation of polyphenolic compounds (flavonols) that includes hemoglobin-containing red blood cells and serum albumin was proposed. The distribution of flavonols between the erythrocyte fraction and albumin was studied. Hemoglobin was shown to play a role in the biotransformation of flavonols. The formation of several intermediate and final products of pseudoperoxidase oxidation of flavonols catalyzed by methemoglobin was established by UV spectrophotometry and RP-HPLC. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 153-157, March-April, 2009.     

Experimental and theoretical investigation effect of flavonols antioxidants on DNA damage

A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0–15.0 and 15.0–500.0 pmol L⁻¹. The detection limit was found 0.5 pmol L⁻¹ and 0.6 pmol L⁻¹ for guanine and adenine, respectively. To confirm the electrochemical results, Uv–Vis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interaction between the antioxidant, AO and DNA.     

Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin

A limitation of large-scale viticultural trials is the time and cost of comprehensive compositional analysis of the fruit by high-performance liquid chromatography (HPLC). In addition, separate methods have generally been required to identify and quantify different classes of metabolites. To address these shortcomings a reversed-phase HPLC method was developed to simultaneously separate the anthocyanins and flavonols present in grape skins. The method employs a methanol and water gradient acidified with 10% formic acid with a run-time of 48 min including re-equilibration. Identity of anthocyanins and flavonols in Shiraz (Vitis vinifera L.) skin was confirmed by mass spectral analysis.     

Mechanistic study on the enzymatic oxidation of flavonols

Several flavonols have been transformed upon treatment by Trametes versicolor laccase. Most of the major oxidation products have been isolated by HPLC as pure compounds and their structures have been, when possible, investigated through spectral methods (HPLC-MS and NMR). The results are coherent with the predominance of a dismutation process, leading to cation formation, over direct radical-radical coupling.     

Simultaneous analysis of anthocyanins and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry

A fast and reliable HPLC method for the simultaneous separation of anthocyanins and flavonols in lotus petals was developed based on the study of four candidate solvent systems. Fifteen flavonoids were identified by high-performance liquid chromatography with photodiode array detection/mass spectrometry. Among them, two anthocyanins and nine flavonols were discovered in lotus petals for the first time. This work is valuable for both the hybrid breeding on lotus oriented to flower color and the utilization of lotus petals as functional food materials.     

Inclusion complexes of modified cyclodextrins with some flavonols

The inclusion complexes of four flavonols with modified cyclodextrins (CDs) have been investigated. The effect of heptakis (2,6-di-O-methyl) β-cyclodextrin (DM-β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on the aqueous solubility of flavonols, namely, galangin, kaempferol, quercetin, and myricetin was investigated, respectively. The increased solubility of all flavonols in the presence of CD was evidenced. The NMR experiment and molecular modeling studies showed that flavonols interact with each modified CD through different binding modes. Flavonols can complex with CDs largely by two binding modes. The first one is that B-ring of flavonols is oriented toward secondary rim of CD. The second one is that A-ring of flavonols is oriented toward secondary rim of CD. Whereas only the first mode was observed in DM-β-CD complexes, both the first and the second mode were observed in HP-β-CD complexes in this study.     

Synthesis of a library of glycosylated flavonols

Flavonols are an important class of natural products isolated from plants. Some glycosylated flavonols showed very interesting biological activities. A library of flavonols has been made through Algar-Flynn-Oyamada reaction from 2′-hydroxyacetophenones and benzaldehydes. Glycosylation of these flavonols with various glycosyl donors affords a library of glycosylated flavonols. These compounds are potentially useful pharmacologically active compounds and will be studied for biological activities.     

Solid-phase extraction procedure for determination of phenolic acids and some flavonols in honey

The solid-phase extraction procedure (SPE) for isolation and preconcentration of phenolic acids (gallic, p-HBA, p-coumaric, vanillic, caffeic and syringic acid) and some flavonols (rutin, quercetin and kaempferol) from honey samples prior to their determination by HPLC is reported. Different solid sorbents such as Bond Elut octadecyl C(18), Oasis HLB, Strata-X and Amberlite XAD-2 were tested for this purpose. The best results were obtain when aqueous solution of honey (100 mL) was adjusted to pH 2 and passed through the microcolumn containing 2.5 g of Oasis HB followed by washing the sorbent with 50 mL of acidified water (pH 2). The analytes were then eluted with methanol. The proposed method permits the quantification of the studied compounds with the limit of detections ranged from 25 ng kg(-1) to 0.75 microg kg(-1) for p-HBA and quercetin, respectively. The precision of the overall analytical procedure was estimated by measuring the within-day repeatability and the relative standard deviations of the parallel (n=3) results were in the range of 1.9-10.1%. The method was tested for real honey samples from different botanical origins.     

A sensitive method for the detection and quantification of ginkgo flavonols from plasma

Extracts from Ginkgo biloba leaves (family Ginkgoaceae) have antioxidant and free radical scavenging effects, largely attributed to the flavonols, which are a major class of functional components in ginkgo extracts. In order to facilitate analysis of systemic exposure to ginkgo-derived products in animals and/or humans, we developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method that is capable of routinely monitoring plasma levels of ginkgo flavonols. We used an initial acidic hydrolysis step to convert the plasma ginkgo flavonol conjugates into their aglycone forms [quercetin (QCT), kaempferol (KMF) and isorhamnetin (ISR)] prior to EtOAc-based extraction and subsequent LC/MS/MS-based analysis. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of HCOOH (0.01 per thousand) dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode; the efficiencies were approximately 4-, approximately 8- and approximately 20-fold higher for QCT, KMF and ISR, respectively, versus the results obtained using an electrolyte-free mobile phase, or approximately 2-, approximately 3- and approximately 4-fold higher, respectively, versus the results obtained using a mobile phase containing the more commonly utilized concentration of HCOOH (1 per thousand). In addition, use of the low concentration of HCOOH also decreased undesired matrix effects. These favorable effects have been referred to as 'LC-electrolyte effects'. Due to structural differences in the B-ring substituent, different types of precursor-to-product ion pairs (m/z 301 --> 151 for QCT, 285 --> 187 for KMF, and 315 --> 300 for ISR) were used for the selected reaction monitoring of the analytes. In addition, the chromatographic conditions were optimized on the basis of an initial scouting of matrix effects on analyte ionization. Despite the absence of an internal standard, the validation results consistently demonstrated that our bioassay is valid, reproducible, and reliable. The newly developed assay provided lower limits of quantification of 1.3, 1.3 and 0.4 pg on-column for QCT, KMF and ISR, respectively, which is more sensitive than any previously reported method for determining ginkgo flavonols. Finally, the assay suitability was demonstrated in a pilot pharmacokinetic measurement of a pharmaceutical ginkgo product in a beagle dog. This newly developed method should prove useful for wide-scale monitoring of ginkgo flavonol plasma concentrations for both pharmaceutical investigations and clinical applications.     

Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection

A novel method based on reversed-phase high-performance liquid chromatography with chemiluminescence detection has been developed for the simultaneous determination of three flavonols including quercetin, kaempferol, and isorhamnetin. The procedure was based on the chemiluminescent enhancement by flavonols of the cerium(IV)-rhodamine 6G system in sulfuric acid medium. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Good separation was achieved with isocratic elution using a mixture of methanol and aqueous 1.0% acetic acid (37:63, v/v) within 25 min. Under optimized conditions, the linear working range covers 3 orders of magnitude with relative standard deviations below 4.5% for 11 replicate injected flavonol samples, and detection limits (S/N= 3) were 1.6 x 10(-8), 3.5 x 10(-9), and 6.5 x 10(-9) g mL(-1) for quercetin, kaempferol, and isorhamnetin, respectively. The chemiluminescence reaction was compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the determination of three active flavonols in phytopharmaceuticals of Hippophae rhamnoides L. After a simple extraction procedure, the repeatability and recovery were satisfactory.     

Antioxidative effects of flavonols and their glycosides against the free-radical-induced peroxidation of linoleic acid in solution and in micelles

The antioxidative effect of flavonols and their glycosides against the peroxidation of linoleic acid has been studied in homogeneous solution (tBuOH/H(2)O, 3:2) and in sodium dodecyl sulfate and cetyl trimethylammonium bromide micelles. The peroxidation was initiated thermally by the water-soluble initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the reaction kinetics were studied by monitoring the formation of linoleic acid hydroperoxides. The synergistic antioxidant effect of the flavonols with alpha-tocopherol (vitamin E) was also studied by following the decay kinetics of alpha-tocopherol and the alpha-tocopheroxyl radical. Kinetic analysis of the antioxidative process demonstrates that the flavonols are effective antioxidants in solution and in micelles, either alone or in combination with alpha-tocopherol. The antioxidative action involves trapping the initiating radicals in solution or in the bulk-water phase of the micelles, trapping the propagating lipid peroxyl radicals on the surface of the micelles, and regenerating alpha-tocopherol by reducing the alpha-tocopheroxyl radical. It was found that the antioxidant activity of the flavonols and their glycosides depends significantly on the position and number of the hydroxy groups, the oxidation potential of the molecule, and the reaction medium. The flavonols bearing ortho-dihydroxy groups possess significantly higher antioxidative activity than those without such functionalities, and the glycosides are less active than their parent aglycones. The activity of the flavonols is higher in micelles than in solution, while the activity of alpha-tocopherol is lower in micelles than in solution. This is because the predominant factor for controlling the activity is the hydrogen-bonding interaction of the antioxidant with the micellar surface in the case of hydrophilic flavonols, while it is the inter- and intramicellar diffusion in the case of lipophilic alpha-tocopherol.     

Carbon-13 NMR studies of flavonoids—I : Flavones and flavonols

¹³C-NMR spectra of hydroxylated flavones and flavonols are presented for the first time. Analyses of the spectra are derived by consideration of a series of acetophenones, cinnamic acids, flavones and flavonols of increasing oxygenation pattern. The accepted substitution additivity rules have been shown to hold for these compounds except in cases where structural modifications involve the C-3,4 and 5 positions.     

Differential pulse polarographic determination of the herbicides atrazine, prometrine and simazine

A differential pulse polarographic method, using the dropping mercury electrode for the determination of the herbicides atrazine, prometrine and simazine is described. The optimum pH is 2. The limit of detection is 8 x 10(-8)M, corresponding to about 15 mu/l. The electrochemical behaviour of the compounds on glassy-carbon and mercury-coated glassy-carbon electrodes was also examined with a view to its use for electrochemical detection of the herbicides after their separation by HPLC.     

Speciation of iron using HPLC with electrochemical and flame-AAS detection

Summary A combined detection system for HPLC is presented, which consists of an electrochemical detector for iron(II) detection and an on-line flame-AAS detector for total iron. Detection limits are 5 ng iron for AAS and 1 ng iron(II) for electrochemical detection. Quantitative analysis of separated iron species is possible, even if iron(II) and iron(III) coelute. The proposed system is used for the investigation of coupled complexation and redox equilibria (e.g. Fe + oxalate). The application to fruit juice and wine is also presented. The predominant species found in apple juice are iron(II)-malate and iron(III)-citrate and in white wine iron(II)- and iron (III)-tartrates.     

Improved high-performance liquid chromatographic method for the determination of coenzyme Q10 in plasma.

Coenzyme (Co) Q10 was dissociated from lipoproteins in plasma by treatment with methanol and extraction with n-hexane. Subsequent clean-up on silica gel and C18 solid-phase extraction cartridges with complete recovery (99 +/- 1.2%) produced a clean extract. High-performance liquid chromatographic (HPLC) separation was performed on a C18 reversed-phase column. Three simple, rapid procedures are presented: HPLC with final UV (275 nm) detection, a microanalysis utilizing a three-electrode electrochemical detector and a microanalysis with column-switching HPLC and electrochemical detection. The methods correlate very well with classical ethanol-n-hexane extraction with UV detection. The identity and purity of the Co Q10 peak were investigated and the resulting methods were concluded to be suitable for total plasma Co Q10 determination. The average level in healthy subjects was 0.80 +/- 0.20 mg/l; the minimum detectable Co Q10 plasma level was 0.05 and 0.005 mg/l for UV and electrochemical detection, respectively. The methods were applied to many samples and the plasma Co Q10 reference values for healthy subjects, athletes, hyperthyroid, hypothyroid and hypercholesterolaemic patients are given.     

Determination of organic acids, amino acids and saccharides by high-performance liquid chromatography and a postcolumn enzyme reactor with amperometric detection.

A technique for the determination of organic acids, amino acids and sugars is described. The compounds of interest are separated by high-performance liquid chromatography (HPLC) and converted on-line by immobilized enzymes. The enzymes employed are covalently bound to a synthetic carrier. Hydrogen peroxide, which is produced in the reaction with oxidases, makes possible the application of an electrochemical detector. This arrangement combines the separation efficiency of HPLC, the substrate specificity of enzymes and the high sensitivity of electrochemical detection. The enzymes act according to known reaction mechanisms, but coupling with HPLC leads to a promising extension in the field of biosensors. The simple pretreatment of the samples (often a dilution step is sufficient) allows a rapid analysis of foodstuffs and biological or clinical extracts. The examples presented demonstrate the very high sensitivity of the method with detection limits in the nano- to picomolar range and a wide field of application.     

Validation of a liquid chromatography ionspray mass spectrometry method for the analysis of flavanones, flavones and flavonols

The application of liquid chromatography/mass spectrometry (LC/MS) with a TurboIonspray (TIS) interface was investigated as a new method for the analysis of flavonoids. Eleven compounds belonging to three different classes of flavonoids were studied: eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin (flavanone glycosides), quercetin, kaempferol, galangin (flavonol aglycones), chrysin, apigenin (flavone aglycones). Chromatographic separations were performed under reversed-phase conditions using a C18 narrow-bore LC column; a mixture of an aqueous solution of formic acid (pH 2.4) and acetonitrile was used as the mobile phase. Isocratic elution was operated in the case of flavanones, whereas gradient elution was used for the simultaneous separation of flavones and flavonols. The adaptability of TIS to high flow applications allows the use of LC eluent flow rates at 200 μL/min without post-column splitting. Qualitative analysis was performed in negative-ion (NI) full-scan mode, whereas response linearity, detection limits and precision of the method were studied under NI selected ion monitoring (SIM) conditions. Characterization of isomers differing in the glycosylation was found to be possible on the basis of different mass spectra. Detection limits in the low-ng range (0.08-0.4 ng) were found, about twenty-fold lower than those reported previously. The method was applied to identify and determine the content of flavonoids in an orange juice sample. Copyright 1999 John Wiley & Sons, Ltd.     

Inhibition of free radical initiated peroxidation of human erythrocyte ghosts by flavonols and their glycosides

The in vitro peroxidation of human erythrocyte ghosts was used as a model to study the free radical-induced damage of biological membranes and the protective effect of flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnopyranoside (QR), rutin (R), morin (MO), kaempferol (K) and kaempferol glucoside (KG). The peroxidation was initiated by a water-soluble free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AMPAD) at physiological temperature, and monitored by oxygen uptake. Kinetic analysis of the peroxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AMPAD-initiated oxidative damage of human erythrocyte ghosts, and that the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities and the glycosides are less active than their parent aglycones.     

Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.