On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Potential of Capillary Electrophoresis for the Profiling of Propolis

The usefulness of capillary electrophoresis (CE) with diode array detection for the profiling of Propolis, a hive product, is investigated. Water extracts of Propolis were analyzed with both capillary zone electrophoresis (CZE) at pH 7.0 and 9.3, and micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate at pH 9.3. Characteristic profiles were obtained and several organic acids and preservatives could be identified by means of library comparison of the recorded UV spectra combined with addition of reference compounds to the extracts. The selectivity of the CZE and MEKC system differed considerably but the information obtained with both methods was similar. The dry residues of the water extraction were extracted with ethanol-water (70 : 30, v/v) and analyzed with the MEKC system to enable the separation of the more hydrophobic constituents of the Propolis samples. Complex profiles containing various well separated peaks were obtained allowing the identification of some interesting flavonoids. On the basis of the recorded CZE and MEKC profiles, the Propolis samples could be divided into two clearly different groups which are probably from a different origin.     

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5%?v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS2 results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2?μg/mL (extracts).     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Capillary electrophoretic separation of phenolic diterpenes from rosemary

The major phenolic diterpenes responsible for the antioxidant properties of rosemary extracts, namely carnosol and carnosic acid, were separated by capillary zone electrophoresis (CZE) using a 56 cm long uncoated fused-silica capillary and a 50 mM disodium tetraborate buffer of pH 10.1. The effect of the buffer type, pH and concentration, and the capillary length on the separation, was studied. Carnosol and carnosic acid were identified in the electrophoregrams of rosemary extracts through their migration times and UV spectra obtained by CZE analysis of pure compounds isolated from a rosemary extract by HPLC fractionation. The CZE method had good reproducibility (relative standard deviation less than 5%) and was applied to compare the contents of carnosol and carnosic acid in solid and oil-dispersed commercial extracts of rosemary and in rosemary leaves. The separation of carnosol and carnosic acid was accomplished in less than 11 min.     

Chromatographic performance of a new polar poly(ethylene glycol) bonded phase for the phytochemical analysis of Hypericum perforatum L

The aim of this study was to evaluate the chromatographic performance of a poly(ethylene glycol) (PEG) stationary phase for the HPLC analysis of the secondary metabolites (chlorogenic acid, flavonoids, phloroglucinols and naphthodianthrones) in methanolic extracts of Hypericum perforatum L. (St. John's Wort) flowering tops, herbal medicinal products and dietary supplements. A fast and reliable method was developed. The analyses were carried out on a Supelco Discovery HS PEG column (150 mm x 4.6 mm i.d., 5 microm). A gradient mobile phase, composed of 0.1 M aqueous acetic acid solution (pH 2.8) and methanol-acetonitrile (5:4, v/v), was used. The flow rate was 1 mL/min. The photodiode array detector monitored the eluent at 270 (for chlorogenic acid, flavonoids and phloroglucinols) and 590 nm (for naphthodianthrones). The column was maintained at room temperature. The total running time was 40 min. The method was validated and showed good linearity, precision, accuracy, sensitivity and specificity. Through the above described phytochemical markers, this technique allowed the unequivocal identification and standardization of H. perforatum plant material and phytoproducts. The quantification data highlighted the fact that the products on sale, in particular those labeled as dietary supplements, varied widely in the quantitative composition of the active constituents. The developed method could be considered suitable for the quality control of H. perforatum herb and derivatives.     

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.     

Determination of flavonoids by high-performance liquid chromatography and capillary electrophoresis

The compounds of flavonoid, an important group in nature, can prevent coronary heart disease and anticancer by virtue of the characteristics of antioxidation. Nine flavonoids most often seen in grape wine, namely apigenin, baicalein, naringenin, luteolin, hesperetin, galangin, kaempferol, quercetin, and myricetine, were determined by means of high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) in this work. A successful resolution was obtained from an unusual additive of tetrahydrofuran in mobile phase by HPLC. One notable thing is that the mixture of luteolin and quercetin could be separated for the first time by HPLC. In addition, the better detection limit was still attainable even with the use of tetrahydrofuran. The detection limits of CZE performed in borate buffer were hundreds-fold better than in previous reports. Furthermore, the retention and migration behavior of the analytes studied were discussed. As the result of this study, the elution order of flavone and flavonone was reversed to the contention proposed by Wulf et al. It was predictable from the interaction with tetrahydrofuran. Consequently, the extracts from grape wine with solid-phase extraction were analyzed by developing methods of HPLC and CZE. The obtained recoveries ranged from 90 to 107% and the relative standard deviations were under 6.3%.     

高效液相色谱法和毛细管电泳法测定甘草制品中甘草酸的含量

分别用高效液相色谱法（ＨＰＬＣ）、毛细管区带电泳法（ＣＺＥ）、胶束电动毛细管色谱法（ＭＥＣＣ）测定了甘草制品中甘草酸的含量。对ＨＰＬＣ,ＣＺＥ,ＭＥＣＣ的分析条件作了一些选择实验,结果表明ＭＥＣＣ法与ＨＰＬＣ法分析数据接近、比较准确,而且前者比ＨＰＬＣ法分离效率高、溶剂用量少,是一种很有发展潜力的分析方法。     

Comparative chemical fingerprinting of Oroxylum indicum and Scutellaria baicalensis using liquid chromatography and mass spectrometry

Introduction: Identification of Oroxylum indicum and Scutellaria baicalensis provides an interesting challenge in selection of biomarker compound to be used in routine analysis. Both plants have similar phytochemical profile and are rich sources of flavones and flavone glycosides. The objective of this study was to prepare the chemical fingerprinting of O. indicum bark and S. baicalensis roots using the liquid chromatography and mass spectroscopy in single chromatographic method. Materials and methods: Extracts prepared using various solvent systems (methanol, aqueous methanol, chloroform, hexane, and water) of both plants were analyzed using C18 reverse phase column with solvent system containing acetonitrile and 0.1% formic acid. Major flavonoids were identified based on mass spectra, fragmentation pattern, and UV spectra. Results: In this article, well-resolved high-performance liquid chromatographic (HPLC) separation in both plant extracts was obtained and chemical fingerprints for both plant extracts were established and flavonoids present (baicalin, oroxylin A-7-O-glucuronide, chrysin-7-O-glucuronide, baicalein, chrysin, oroxylin-A, wogonin, skullcap flavone II) were identified as possible biomarkers. Conclusion: Mass spectrometry coupled with HPLC can be a tool for fingerprinting of various natural products used in dietary supplement industry. The fingerprint developed in the article can be used for quality evaluation as well as identifying possible adulteration of extracts of both the plants.     

基于高效液相色谱定量指纹图谱和液相色谱-质谱联用定量的酸枣仁提取物质量考察

酸枣仁具有显著的改善睡眠和抗焦虑等作用,其提取物在助眠类功能食品开发中应用前景广阔。但目前市场上酸枣仁提取物质量参差不齐,缺乏统一标准,企业在使用时面临较大的质量风险,因此亟须建立一种准确、全面的内控质量评价方法。针对酸枣仁提取物中黄酮和皂苷两类主要活性成分紫外响应差异巨大且水提物中皂苷成分含量低的问题,该研究建立了酸枣仁水提物HPLC定量指纹图谱方法,共标定了8个共有峰。通过对照品指认、文献比对以及高效液相色谱-四极杆飞行时间质谱数据解析,8个共有峰均为黄酮类化合物,该方法可同时实现7种黄酮成分的半定量对比分析和斯皮诺素的含量测定;采用超高效液相色谱-三重四极杆质谱,在正离子模式下,以多反应监测扫描方式可实现酸枣仁皂苷A和B的含量测定;最终以雷达图展现上述10种成分的半定量和定量数据。应用上述方法,该研究对比分析了实验室自制的3批酸枣仁水提物和15家供应商的15批提取物样品。结果显示,实验室自制的3批酸枣仁水提物虽然原料来自不同饮片企业,但总体差异性不大,而不同厂家提供的酸枣仁提取物样品成分含量差异巨大,提示不同厂家存在辅料稀释、理枣仁掺假和醇提或纯化富集等情况。该法为企业制定内控质量标准和筛选合格供应商提供了依据。     

Preparative isolation and purification of three flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai was successfully established by using chloroform-methanol-water (4:3.5:2, v/v) as the two-phase solvent system. The method yielded 11.4 mg of epimedokoreanoside I, 46.5 mg of icariin and 17.7 mg of icariside II from 200 mg of the crude sample in one-step separation with the purity of 98.2%, 99.7% and 98.5%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of the flavonoids were identified by 1H NMR and 13C NMR.     

Quantification of flavonoid glycosides in an aqueous extract from the traditional Mongolian medicinal plant Dianthus versicolor FISCH

An HPLC‐diode array detection (DAD) method was established in order to investigate dried aerial parts of Dianthus versicolor FISCH. (Caryophyllaceae), a plant used in traditional Mongolian medicine against liver impairment. Aqueous extracts were separated on an Aquasil^® C₁₈ column with a linear gradient of acetonitrile (ACN) and water (adjusted to pH 2.8 with formic acid) as the mobile phase. LC‐IT‐MS facilitated the assignment of 26 flavonoids, among them a series of rare C‐glycosylated as well as O‐glycosylated derivatives, which are assumed to be the active principles. Quantification was performed and validated using isovitexin‐7‐O‐glucoside (saponarin) as the external standard. The method showed good linear behaviour (r² ≥0.9999) over the investigated concentration range (0.007–3.5 mg/mL). The good precision of the method allowed the successful qualitative and quantitative analysis of flavonoid‐glycosides in the aqueous extracts prepared from five different D. versicolor samples. Depending on the origin of the samples, the total flavonoid content was found to vary considerably from 0.41 to 3.30% in the aqueous extracts and from 0.07 to 0.57% in the crude drug. In addition, the relative composition of the various flavonoids was found to differ strongly. These results highlight the need for proper quality control for this herbal drug.     

High performance liquid chromatography/electrospray mass spectrometry of Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) is a widely distributed herbaceous perennial plant which has been well known as a medicinal plant since antiquity. In recent years, H. perforatum has received increasing attention for the treatment of depression and other neuralgic disorders. The main constituents of H. perforatum extract include flavonoids, naphthodianthrones, phloroglucinols, essential oils and xanthones. The present work reports the analysis of naphthodianthrones and phloroglucinols in H. perforatum extracts by means of high performance liquid chromatography (HPLC) coupled simultaneously to a diode array detector (DAD) and electrospray mass spectrometry (ESI-MS). Hypericin, pseudohypericin, hyperforin and adhyperforin were separated and identified on the base of their on-line UV and mass spectra. Quantitative analysis of hypericin derivatives in different extracts of H. perforatum using DAD and MS detectors was performed. In addition, direct infusion ESI-MS of H. perforatum extracts was applied to obtain rapid mass fingerprints of constituents present in the sample.     

Analysis of Carbaryl and carbofuran in tobacco samples by HRGC,HPLC, and CZE

Determination of carbamate residues in tobacco samples was carried out by three different methods, two having been described in the literature and one developed for the purpose of this work. The extracts were analyzed by HRGC-FID (using derivatization), by HPLC-RP with photodiode array detection and by Capillary Zone Electrophoresis (CZE) with UV detection. The extraction method developed used in conjunction with capillary zone electrophoresis showed to be the most suitable for carbamate residue analysis in real tobacco samples. The developed method presented better recovery results in relation to the others. CZE allowed the determination of carbamate residue in real tobacco sample while HPLC and HRGC could not do this, due to co-elution problems.     

Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods

The Polynesian medicinal fern Microsorum membranifolium contains very large amounts of ecdysteroids, including ecdysone, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. It also contains large amounts of unusual ecdysteroids which have been unambiguously identified by mass spectrometry and nuclear magnetic resonance. A new class of ecdysteroid conjugates (3-glucosyl-ferulates of 2-deoxyecdysone and 2-deoxy-20-hydroxyecdysone) is isolated, together with a new glycoside (2-deoxyecdysone 25-rhamnoside). The simultaneous presence of a sugar and an aromatic moiety results in a very particular chromatographic behavior of these conjugates. They behave like flavonoids and polyphenols when using the classical purification on polyamide, aimed at removing the latter from crude plant extracts, and would therefore be lost. They elute as non-polar ecdysteroids on reversed-phase high-performance liquid chromatography (RP-HPLC), whereas their behavior on normal-phase (NP) HPLC is strongly dependent on the mobile phase composition. Our data highlight the importance of selectivity in the choice of HPLC methods used for ecdysteroid separations.     

Characterization of the methanolic extract of hops using capillary electrophoresis-electrospray ionization-mass spectrometry

Hops are used almost exclusively for bitterness and flavor by brewers. We propose the first analytical application of CZE coupled to ESI-MS for the separation and structural elucidation of organic compounds in the methanolic extracts of hops, and different extraction procedures of the plant material have been carried out. The proposed method permits the identification of hop polyphenols (flavonoids glycosides and chalcones), bitter acids (alpha-acids and beta-acids), and their oxidation products. The optimization of CZE parameters (pH, concentration, and type of buffer) and ESI-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure, drying gas flow rate, temperature, and compound stability) have permitted the development of a rapid, simple, direct, and straightforward CZE-ESI-MS method for the identification of components of methanolic extracts from different hops used in the brewing process.     

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.     

RP-HPTLC fingerprinting of secondary metabolites from Nephrolepis exaltata and Cycas revoluta

This study aims to develop a quantitative fingerprinting between two evolutionary close plant species Nephrolepis exaltata (L.) Schott and Cycas revoluta Thunb. The crude plant extracts were prepared in 1:1:1, v/v/v: Ethyl Acetate: n-Hexane: Methanol (AR) solvent system through cold extraction for 24 Hrs. The plant extracts were diluted (10 mg/mL) with a similar solvent system spotted (10 μL) on silica gel 60 F²⁵⁴ thin-layer chromatography plates. Five mobile phases (i) tetrahydrofuran (THF): toluene: formic acid: water (16:8:2:1) (ii) toluene: ethyl acetate: diethylamine (7:2:1) (iii) toluene: ethyl acetate: formic acid (7:3:0.1) (iv) n-hexane: ethyl acetate (7.2:2.9) (v) toluene: methanol (9:1) were used. The results were scanned at 254 nm, 366 nm, and in visible white light. This study gave minimum compact spots between Rf 0.06 to 0.051 corresponding to terpenoids, simultaneously for flavonoids spots were found between Rf 0.004 to 0.910. Total eight phenolic bands were found in Cycas revoluta among which five was found in leaf extract with Max RF of 0.033, 0.093, 0.273, 0.901 and 0.964 whereas three bands were found in stem extract with Max Rf of 0.007, 0.897 and 0.954. The presence of fifteen different types of flavonoids was determined by the flavonoids profile at Rf 0.004, 0.027, 0.047, 0.073, 0.134, 0.230, 0.303, 0.369, 0.369, 0.427, 0.514, 0.631, 0.703, 0.757, 0.846 and 0.910 for Nephrolepis exaltata. HPTLC fingerprint has shown several peaks with different Rf for different mobile phases. The fingerprinting would be helpful in the identification, and authentication of these species ecologically and therapeutically.     

HPLC/DAD,Antibacterial and Antioxidant Activities of Plectranthus Species (Lamiaceae) Combined with the Chemometric Calculations

The increase in antibiotic resistance and the emergence of new bacterial infections have intensified the research for natural products from plants with associated therapy. This study aimed to verify the antibacterial and antioxidant activity of crude extracts of the genus Plectranthus species, being the first report on the modulation of aminoglycosides antibiotic activity by Plectranthus amboinicus extracts. The chemical composition was obtained by chemical prospecting and High-Performance Liquid Chromatography with diode arrangement detector (HPLC/DAD). The antibacterial activities of the extracts alone or in association with aminoglycosides were analyzed using the microdilution test. The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging. The phytochemical prospection allowed the flavonoids, saponins, tannins and triterpenoids to be identified. Quercetin, rutin, gallic acid, chlorogenic acid, caffeic acid, catechin, kaempferol, glycosylated kaempferol, quercitrin, and isoquercitrin were identified and quantified. The principal component analysis (PCA) observed the influence of flavonoids and phenolic acids from Plectranthus species on studied activities. Phytochemical tests with the extracts indicated, especially, the presence of flavonoids, confirmed by quantitative analysis by HPLC. The results revealed antibacterial activities, and synergistic effects combined with aminoglycosides, as well as antioxidant potential, especially for P. ornatus species, with IC₅₀ of 32.21 µg/mL. Multivariate analyzes show that the inclusion of data from the antioxidant and antibacterial activity suggests that the antioxidant effect of these species presents a significant contribution to the synergistic effect of phytoconstituents, especially based on the flavonoid contents. The results of this study suggest the antibacterial activity of Plectranthus extracts, as well as their potential in modifying the resistance of the analyzed aminoglycosides.