Development of an on-line lab-on-valve micro-solid phase extraction system coupled to liquid chromatography for the determination of flavonoids in citrus juices.

An on-line lab-on-valve system for renewable micro-solid phase extraction coupled with high performance liquid chromatography with photodiode array detection was developed for the determination of hesperidin and diosmin in natural and commercial citrus samples. The method is based on the automatic loading of 8 mL of sample into the C18 resin, followed by matrix clean-up and elution of the analytes with 0.27 mL of acetonitrile. Next, 0.02 mL of the extract were injected into the chromatographic system. Separation was performed by using a Symmetry® C18 column (250 × 3.0 mm x 5.0 µm) with a mobile phase consisting in a mixture of methanol, acetonitrile and acidic water (pH = 2.5) in gradient mode at 0.58 mL min-1. The detection wavelength was chosen as 285 nm. Limits of detection, quantification and relative standard deviations were lower than 0.1 µg mL-1, 0.3 µg mL-1 and 4.1%, respectively.

NIR spectroscopy-multivariate analysis for discrimination and bioactive compounds prediction of different Citrus species peels.

Near Infrared (NIR) method combined with chemometrics was utilized to achieve the target of deeper insight into the chemical diversity and to discriminate the different species and chemovarieties of Citrus peels. Unsupervised principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used for comparing of samples. A clear separation among the eight investigated species and cultivars was revealed, except for the red and white C. paradisi peels samples. Furthermore, fingerprint-bioflavonoids content relationship was modeled by partial least squares regression. A practical approach based on reflectance NIR measurements and partial least squares regression (PLSR) was demonstrated for quantitative determination of the bioflavonoids hesperidin and diosmin and compared to other reported methods. The regression coefficients (R2) between predicted values and pre-determined hesperidin and diosmin content were >0.98, indicating the possibility to simultaneously quantify hesperidin and diosmin in Citrus samples directly from NIR measurements using an adequate PLS regression. Citrus sinensis followed by Citrus reticulata samples were found the most enriched in the bioflavonoids hesperidin and diosmin. NIR-multivariate analysis can therefore be used for discrimination of different varieties and selection of citrus species with desired amounts of specific bioflavonoids which could successfully be analyzed in such complex plant matrices which can prove useful for further pharmaceutical implementation.

A comparative study of Mentha arvensis L. and Mentha haplocalyx Briq. by HPLC.

We have developed a new method to simultaneously determine five marker compounds in Menthae Herba via HPLC/PDA - including hesperidin (1), rosmarinic acid (2), diosmin (3), didymin (4) and buddleoside (5). The newly developed method was successfully used to analyse for two species (Mentha arvensis L. and Mentha haplocalyx Briq.) of Menthae Herba, and the satisfactory results were obtained from the validation of developed method. The pattern analysis could greatly discriminate between M. arvensis L. and M. haplocalyx Briq. In conclusion, the proposed HPLC/PDA method is suitable for quality evaluation of Menthae Herba.

Use of multiresponse statistical techniques to optimize the separation of diosmin, hesperidin, diosmetin and hesperitin in different pharmaceutical preparations by high performance liquid chromatography with UV-DAD.

A new method for the separation and determination of four flavonoids: hesperidin (HES), diosmin (DIO), hesperitin (HTIN), and diosmetin (DTIN) in pure form and pharmaceutical formulations has been developed by using high performance liquid chromatography (HPLC) with UV-DAD detection. Multivariate statistics (2k full factorial and Box Behnken Designs) has been used for the multiresponse optimization of the chromatographic separation, which was completed in 22min, and carried out on a symmetry® C18 column (250×3mm; 5µm) as stationary phase. Separation was conducted by gradient elution mode using a mixture of methanol, acetonitrile and water pH: 2.5 (CH3COOH), as mobile phase. Analytes were separated setting the column at 22°C, with a flow rate of 0.58mLmin-1 and detected at 285nm. Under the optimized conditions, the flavonoids showed retention times of: 8.62, 11.53, 18.55 and 19.94min for HES, DIO, HTIN and DTIN, respectively. Limits of detection and quantification were <0.0156µgmL-1 and <0.100µgmL-1, respectively. Linearity was achieved with good correlation coefficients values (r2=0.999; n=5). Intra-day and inter-day precision were found to be less than 3.44% (n=7). Finally, the proposed method was successfully applied to determine the target flavonoids in pharmaceutical preparations with satisfactory recoveries (between 95.2% and 107.9%), demonstrating that should also find application in the quality control, as well as in the pharmacokinetic studies of these drugs.

Diosmin--isolation techniques, determination in plant material and pharmaceutical formulations, and clinical use.

Diosmin is a naturally occurring flavone glycoside used in the treatment of venous diseases. In this review, we present the clinical aspects of the use of diosmin preparations in venous stasis, hemorrheologic disorders and vein wall remodeling. Because of its multiple applications in biology and its many therapeutic activities, research on isolation and identification of diosmin is of high relevance. The aim of this review is to present an overview of techniques of isolation and separation of diosmin in plant material, pharmaceutical formulations such as Daflon, Diosed and Dioven tablets, and biological fluids.

HPLC determination of hesperidin, diosmin and eriocitrin in Iranian lime juice using polyamide as an adsorbent for solid phase extraction.

Solid phase extraction (SPE) and HPLC were used for simultaneous determination of hesperidin, diosmin and eriocitrin in Iranian lime juice samples. The method involved very simple efficient SPE with polyamide cartridge, the use of mixture of water/acetonitrile/acetic acid (78:19:3, v/v) as a mobile phase at a flow rate of 0.8 mL/min and UV detection at 280 nm. Optimum conditions for SPE were achieved using 8 mL water/methanol (85:15, v/v, pH=3) as the washing solution and 4 mL methanol for elution. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Good clean-up and high>90% were observed for all analytes. Limits of detection, limits of quantification, linear range, recovery, repeatability of retention times, and peak areas for hesperidin, diosmin and eriocitrin were 0.0283-0.0512 µg/mL, 0.0857-0.155 µg/mL, 0.0283-105.0 µg/mL (R² > 0.99), 93.3-98.1%, 3.2-4.7% and 2.8-3.6%, respectively. The method was applied to analysis of lime juice samples obtained from different locations of Iran.

The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin.

The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH(*) assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios.

Two-dimensional liquid chromatography coupled with mass spectrometry for the analysis of Lobelia chinensis Lour. using an ESI/APCI multimode ion source.

A comprehensive approach for the separation and identification of components in a traditional Chinese medicine Lobelia chinensis Lour. was developed using 2D-HPLC coupled with an online photodiode array (PDA) detector and a mass spectrometer. The extract of L. chinensis Lour. was separated on a CN column in the first-dimensional HPLC, and then each of the collected fractions was further separated on an ODS column followed by an online PDA detector. After separation in the two different chromatographic modes, the eluents were delivered to a quadrupole mass spectrometer equipped with a multimode ion source of an ESI and an atmospheric pressure chemical ionization (ESI/APCI). At least 536 components in L. chinensis Lour. extract were detected and 6 of them were identified as apigenin 7-O-rutinoside, luteolin, lobetyolinin, lobetyolin, diosmin, and linarin, respectively, according to their UV spectrum and mass spectrum. The results demonstrated the powerful resolution, high peak capacity, as well as the identification capability of the 2D-HPLC combined with PDA and ESI/APCI-MS for the analyses of complex samples.

Determination of trace levels of diosmin in a pharmaceutical preparation by adsorptive stripping voltammetry at a glassy carbon electrode.

A systematic study on the electrochemical behavior of diosmin in Britton-Robinson buffer (pH 2.0-10.0) at a glassy carbon electrode (GCE) was made. The oxidation process of the drug was found to be quasi-reversible with an adsorption-controlled step. The adsorption stripping response was evaluated with respect to various experimental conditions, such as the pH of the supporting electrolyte, the accumulation potential and the accumulation time. The observed anodic peak current at +0.73 V vs. Ag/AgCl reference electrode increased linearly over two orders of magnitude from 5.0x10(-8) M to 9.0x10(-6) M. A limit of detection down to 3.5x10(-8) M of diosmin at the GCE was achieved with a mean recovery of 97+/-2.1%. Based on the electrochemical data, an open-circuit accumulation step in a stirred sample solution of BR at pH 3.0 was developed. The proposed method was successfully applied to the determination of the drug in pharmaceutical formulations. The results compared favorably with the data obtained via spectrophotometric and HPLC methods.

Determination of flavonoids in citrus juices by micro-HPLC-ESI/MS.

Flavonoids are a large class of naturally occurring aromatic secondary plant metabolites. They constitute one of the most characteristic classes found in nature and more than 4000 flavonoids have been identified and divided into several subclasses. Flavonoids have several effects on human health, mainly related to their antioxidant activity. As a result of the increased interest in the biologically active compounds in food, many research studies have investigated the detection and quantification of antioxidants. Citrus plants are of great interest since they accumulate large amounts of flavonoids and fruit and juices of these species are consumed in large quantities. Despite the extensive research performed on Citrus flavonoids, many compounds still remain unidentified. In this work, flavonoids of five citrus juices: lemon, grapefruit, bergamot, orange, and mandarin were analyzed by microHPLC coupled on-line with an MS detector equipped with an ESI source operating in negative mode. The use of microcolumn HPLC greatly enhanced detection performance. In total, 20 flavonoids of two different subclasses (flavanone and flavone glycosides) were identified with the help of retention data and MS spectral information.

Simultaneous reversed-phase high-performance liquid chromatographic method for the determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical formulations.

Diosmin, hesperidin and naringin are flavonoid glycosides that occur naturally in citrus fruits. They exert a variety of pharmacological properties such as anti-inflammatory, antioxidant and free radical scavenging and antiulcer effects and also inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical preparations. Diosmin, hesperidin, naringin and the internal standard rhoifolin were separated using tetrahydrofuran/water/acetic acid (21:77:2, v/v/v) as the mobile phase at 34 degrees C, using a C8 reversed-phase column. The method was linear in the 0.25-20.0 microg/ml concentration range for all three flavonoid glycosides (r>0.999). The method has been successfully applied to the determination of all three flavonoid glycosides in several samples of different citrus fruit juices sold in Greece and for the determination of diosmin and hesperidin in pharmaceutical preparations.

Flavonoids detection by HPLC-DAD-MS-MS in lemon juices from Sicilian cultivars.

High-performance liquid chromatography-diode array detector (HPLC-DAD) coupled with electronspray mass spectrometry (ESI-MS) was used to detect the flavonoid profile in lemon juices obtained from the main Sicilian cultivars (Femminello comune, Monachello, and Interdonato). Significant amounts of an unusual constituent were found in the lemon juice of the above-mentioned cultivars together with eriocitrin, hesperidin, and diosmin. Following purification by preparative HPLC, the structure was assigned as 6,8-di-C-glucopyranosyldiosmetin by means of DAD-UV, ESI-MS-MS, and (1)H NMR analysis. Three other minor components were also detected. One of these presented a flavone nature, and spectral data and literature references both suggested a 6,8-di-C-glucopyranosylapigenin structure. The different contents of eriocitrin, hesperidin, diosmin, and above minor components in the cultivars allow juices to be readily differentiated.

Improved LC methods for the determination of diosmin and/or hesperidin in plant extracts and pharmaceutical formulations.

Two improved, simple, selective and sensitive reversed-phase HPLC methods were developed for the determination of diosmin and hesperidin simultaneously or separately, using external or internal standard techniques, respectively. The methods were applied for determination of the diosmin and/or hesperidin in Buchu leaves and pharmaceutical dosage forms (three tablets). The methods involved very simple efficient extraction procedures, the use of a LiChrosorb RP-18 column, a mixture of methanol-water (60:40, v/v) as a mobile phase in an isocratic mode at a flow rate of 1.5 ml/min and UV detection at 345 nm. In all HPLC analysis the relative standard deviation did not exceed 1.9%. As low as 50 and 25 ng (per injection) could be detected in less than 6 min with a high degree of accuracy. This is the first report for the determination of the two flavonoids simultaneously in Buchu leaf and tablets containing them.

Selection of hybrids and edible citrus species with a high content in the diosmin functional compound. Modulating effect of plant growth regulators on contents.

The purpose of this study is to identify species, hybrids, and cultivars of edible Citrus species with high contents of diosmin as a functional compound and also to identify the developmental progress of the fruit in which it reaches maximum levels; these findings would be useful for extraction purposes and for the modulating effect of plant growth regulators on diosmin content to increase the level of this flavone. The results obtained reveal that the highest contents of diosmin are present in immature fruits of certain varieties of citron (Buda's finger) and lemon (Meyer), whereas the contents in the edible parts of the fruits are irrelevant from a pharmacological point of view. Similarly, it is shown that it is possible to increase the content of this flavone using hormonal treatments (6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid) during the early stages of fruit growth.