Determination of Five Polyphenols by HPLC/DAD and Discrimination of Apple Varieties

The objective of this study was to set up a method to detect five compounds in fresh smashed apples by HPLC/DAD simultaneously. Different methods have been tested to control browning and ascorbic acid with ultrasonication was adopted. Methanol–water–acetic acid (30:69:1, v/v) containing 2.0 g of ascorbic acid L^?1 was chosen as the extract solvent. The method effectively simplified the sample treatment compared with the traditional ways. And primarily, the results were used to identify between different varieties. The chromatographic separation was performed on an Atlantis C18 (250 mm × 4.5 mm, particle size 5 μm) with a gradient elution program using a mixture of acetonitrile and 2% aqueous acetic acid (v/v) as mobile phase within 20 min at 270 nm wavelength. The variation of the content of five compounds was gallic acid (ND ~1.81 μg g^?1), protocatechuic acid (ND ~1.79 μg g^?1), chlorogenic acid (13.81–189.4 μg g^?1), caffeic acid (6.82–45.02 μg g^?1) and rutin (0.96–18.55 μg g^?1). The results could successfully be used to discriminate between different apple varieties (Gala, Fuji, Delicious, 8th Apple US, Golden Apple, Green Apple and Red Rose); chlorogenic acid and rutin being the polyphenols that contribute most to the differentiation.     

Study on the determination of polyphenols in tobacco by HPLC coupled with ESI-MS after solid-phase extraction

A high-performance liquid chromatography method coupled with electrospray ionization-mass spectrometry for the determination of polyphenols in tobacco is studied. The polyphenols are extracted from a tobacco sample by being refluxed in a boiling water bath with 80% methanol and purified by solid-phase extraction with a C18 cartridge. The chlorogenic acid, rutin, scopoletin, caffeic acid, scopolin, and other polyphenols are satisfactorily separated on a Nova-Pak C18 chromatographic column (3.9 x 150 mm) with methanol and 0.05 mol/L potassium dihydrogen phosphate buffer solution gradient elution as mobile phase at a flow rate of 0.5 mL/min. Each of the polyphenols is monitored by photodiode array detector at its maximum wavelength: chlorogenic acid, 326.1 nm; rutin, 354.8 nm; scopoletin, 344.0 nm; caffeic acid, 323.7 nm; and scopolin, 365.2 nm. The limits of detection are: 100 ng/mL for chlorogenic acid, 125 ng/mL for rutin, 60 ng/mL for scopoletin, 50 ng/mL for caffeic acid, and 100 ng/mL for scopolin. The key polyphenols in tobacco are identified by comparing the retention time, the UV-spectrum, and the mass spectra with those of the standards. The recovery of tobacco polyphenols is 94-105%, and the relative standard deviations are 1.28-1.49%. This method is successfully applied to qualitatively and quantitatively analyze the polyphenols in tobacco with good results.     

Simultaneous Analysis of Sugars and Polyphenols in Tobacco by CZE with Amperometric Detection Based on a Cu2O/MWCNT-COOH Composite Electrode

A composite electrode was fabricated from Cu₂O powder, carboxyl-functionalized multi-wall carbon nanotubes (MWCNT-COOH), and paraffin oil in the proportions 51:17:32 (w/w). This composite electrode was used for amperometric detection (CZE–AD) in simultaneous capillary zone electrophoretic analysis of chlorogenic acid, rutin, sucrose, glucose, mannose, and fructose in tobacco samples. Under the optimum conditions, the six analytes could be separated in 100 mmol L^?1 NaOH buffer within 30 min. Good linearity was achieved in the range 1 × 10^?7–1 × 10^?4 mol L^?1 for the two polyphenols and 5 × 10^?6–1 × 10^?3 mol L^?1 for the four sugars. The detection limits (S/N = 3) for the polyphenols and sugars were as low as 10^?8 mol L^?1 and 10^?6 mol L^?1, respectively.     

Surfactant-assisted dispersive liquid–liquid micro-extraction combined with magnetic solid-phase extraction for analysis of polyphenols in tobacco samples

A novel and simple two-step micro-extraction technique combining surfactant-assisted dispersive liquid–liquid micro-extraction and magnetic solid-phase extraction prior to high-performance liquid chromatography was established for analysis of polyphenols including chlorogenic acid, caffeic acid, and scopoletin in tobacco samples. In the developed system, Fe₃O₄ nanoparticles were synthesized by a one-step chemical co-precipitation method and used to remove hydrophobic substances in tobacco samples by physical adsorption. Low-density solvent (1-heptanol) and cationic surfactant cethyltrimethyl ammonium bromide were employed as extraction solvent and disperser agent, respectively. Under the optimized experimental conditions, a good linearity of the method was obtained over the concentration range from 0.1 to 1000 ng mL^?1 for target analytes. The limits of detection (S/N?=?3) were 0.05 ng mL^?1 for CGA, 0.10 ng mL^?1 for CFA, and 0.12 ng mL^?1 for SP, respectively. Finally, the applicability of the developed method was evaluated by extraction and determination of these three phenolic compounds in tobacco samples and satisfactory average recoveries of spiked samples were between 96.6 and 102.7%.     

固相萃取-高效液相色谱法测定烟草样品中主要的植物多酚

 建立了先用体积分数为 80 %的甲醇水溶液回流提取烟草样品中的植物多酚 ,再采用Sep Pak C18固相微萃取小柱预分离脱脂的前处理方法 ,并选择了合适的分析烟草样品中植物多酚的高效液相色谱条件。采用C18柱 ,甲醇 磷酸二氢钾缓冲液为流动相进行梯度洗脱 ,烟草中主要的植物多酚均达到基线分离。提取各组分在其最大波长下的色谱图 ,根据其峰面积定量 ,并用紫外光谱图对烟草中主要的植物多酚进行辅助定性。在该方法中 ,多酚的标准回收率为 94%～ 10 5 %,RSD为 1 2 8%～ 1 49%。该方法可用于烟草样品中的植物多酚的测定。     

Liquid Chromatographic Analysis of Phenolic Compounds in Organically and Conventionally Grown Varieties of Sour Cherries

Liquid chromatographic analysis of the polyphenols in sour cherries was achieved on bi-functional RP columns with a gradient prepared from methanol and aqueous formic acid. For quantitative analysis the compounds were detected at 320 nm. The method was validated for linearity, precision, and recovery. Chlorogenic acid, neochlorogenic acid, and rutin were detected in sour cherry cultivars. Amounts of neochlorogenic acid were in the range 471–590 μg g⁻¹. For the three sour cherry cultivars investigated in this study there was no significant difference between the chlorogenic acid and rutin content of cherries produced by conventional and organic farming. There were, however, significant differences between the amounts of these compounds in cherries harvested in different years.

     

Simultaneous Determination of Five Phenolic Compounds in Dried Flowers by LC Using DAD Combined Electrochemical Detection

A simple, sensitive and accurate method for the simultaneous separation and determination of apigenin and four phenolic acids including chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid in four dried flowers by high performance liquid chromatography with electrochemical detection (ECD) and diode array detection (DAD) has been established. The detection limits of caffeic acid, p-coumaric acid and ferulic acid obtained with ECD were 3, 1 and 4 ng mL^?1, and LOD of apigenin and chlorogenic acid obtained with DAD were 1 × 10^?2 and 6 × 10^?2 μg mL^?1. The detection and quantification limits of three phenolic compounds obtained with ECD were two to ninefold greater than those obtained with DAD. As electrochemically inactive compounds, apigenin and chlorogenic acid were detected by DAD. All calibration curves showed good linearity (r ≥ 0.9992) within the test ranges. The recoveries ranged from 95.3 to 101.4% (RSD ≤ 2.9%). This approach could provide scientific evidence for comprehensive evaluation about the effect of the medicine and ensure nutrient status of dried flowers.     

Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL^?1 for ascorbic acid, 0.084–0.250 mg mL^?1 for chlorogenic acid, 0.053–0.360 mg mL^?1 for caffeic acid, and 0.016–0.250 mg mL^?1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL^?1 for ascorbic acid, 18 and 54 μg mL^?1 for chlorogenic acid, 37 and 112 μg mL^?1 for caffeic acid, and 11 and 34 μg mL^?1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.     

Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.     

Extraction and Hydrolysis Parameters for Determination of Quercetin in Hypericum perforatum

The optimum conditions for extraction of rutin and quercetin from Hypericum perforatum were investigated. The best efficiency of extraction was achieved with aqueous methanol 40–80% (v/v). For quercetin analysis as aglycone the effect of acid concentration and hydrolysis time on the extraction recovery were also studied. Hydrolysis for 5 min in the presence of 2.8 mol L^?1 HCl as well as for 10 min with 1.1 mol L^?1 HCl efficiently released quercetin from rutin. The content of quercetin?3-O-glycosides (rutin, hyperoside and quercetrin) and quercetin aglycone as well as chlorogenic and caffeic acids in H. perforatum leaves and flowers were determined by HPLC with photodiode-array detection and confirmed by electrospray mass spectrometry.     

高效液相色谱-分段检测法同时测定烟草中14种多酚类化合物

利用高效液相色谱-二极管阵列检测器(HPLC-DAD),建立了同时分析烟草中新绿原酸、绿原酸、隐绿原酸、东莨菪苷、咖啡酸、七叶亭、对香豆酸、阿魏酸、莨菪亭、7-羟基香豆素、芦丁、槲皮苷、山奈酚-3-O-芸香糖苷和槲皮素14种多酚化合物的方法。称取0.02 g烟末,加入5 mL 50%甲醇,超声萃取30 min,取上清液过水相滤膜后,采用Agilent SB-C18柱(3.0 mm×150 mm,1.8μm)进行分离,以0.05 mol/L磷酸二氢钾水溶液和甲醇为流动相,采用分段式检测,获得14种多酚类化合物在各自最佳检测波长下的信号,实现了良好分离。结果表明,14种多酚类化合物的色谱峰面积与其质量浓度呈良好的线性关系,相关系数r2>0.9997,检出限为0.01~0.15μg/mL,定量下限为0.03~0.50μg/mL,日内相对标准偏差(RSD)为0.70%~3.6%,日间RSD为4.0%~6.4%,加标回收率为95.2%~109%。该方法具有良好的灵敏度、精密度和回收率,适合于烟草样品中多种多酚类化合物的同时测定。     

Stepwise Injection Spectrophotometric Determination of Flavonoids in Medicinal Plants

A simple, rapid, and automated method for the spectrophotometric determination of flavonoids in medicinal plants was developed using a stepwise injection manifold. The determination was based on formation of colored complexes of flavonoids with Al(III) in micellar media. Analytical characteristics of the determination were significantly improved when cetylpyridinium chloride was used as a micellar catalyst. It was found that the rate of colored complex formation of rutin with Al(III) increased (k = (1.8 ± 0.1) · 10⁴ min^?1mol^?2 L²) in the presence of cetylpyridinium chloride. Under the optimum conditions, a linear response was found from 0.004 to 0.2% (volume of weight %). The detection limit (3s) was determined as 0.001% rutin versus the weight of the sample. The developed method was used for the analysis of different medicinal plants for flavonoids.     

Large-Volume Sample Stacking with Polarity Switching in CE for Determination of Natural Polyphenols in Plant Extracts

A simple on-column preconcentration method for capillary electrophoretic determination of eight polyphenolic compounds (carnosic acid, cinnamic acid, caffeic acid and rosmarinic acid, quercetin, apigenin, luteolin and rutin) was devised. The method was applied for the assay of polyphenols in methanolic extract of the medicinal plant Orthosiphon Stamineus Benth. The analysis was carried out in fused silica capillaries (I.D. 50 μm, effective length 50 cm, total length 60 cm) with UV detection at 200 nm. The background electrolyte was 50 mM sodium tetraborate of pH 9.0 (adjusted with phosphoric acid). Large volume sample stacking with polarity switching was used for sensitivity enhancement. With sample injection representing 50% of capillary volume and polarity switching at 1.6 min, an average 90-fold enhancement of absorbance signal of the analytes was achieved. The calibration curves were linear (r = 0.9956–0.9994) in the range 0.2 to 1.8 μg mL^?1 of an analyte. The repeatability of migration times and peak areas was characterized by RSD values 0.11–0.57 and 1.63–5.66%, respectively. The proposed method offers favourable limits of detection (9–16 ng mL^?1) that compare well with those of LC.     

Determination of Polyphenols in Lycium barbarum Leaves by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

High-performance liquid chromatography coupled with high-resolution mass spectrometry was used for the determination of polyphenols in Lycium barbarum leaves. Twenty compounds extracted by methanol–water were tentatively identified that included chlorogenic acids, flavonoids, and phenolic acids. Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were identified in Chinese cultivated L. barbarum leaves for the first time. Caffeic acid and isoquercitrin were also present. The concentrations of these compounds in L. barbarum leaves were determined. The results showed that all analytes had linear calibration relationships with limits of detection from 0.318 to 3.35?ng mL^?1. The polyphenols and flavonoids in L. barbarum leaves provided strong 2,2-diphenyl-1-picrylhydrazyl radical-scavenging capacity (IC₅₀ of 23.1?±?0.4 to 26.0?±?0.4?µg mL^?1). This method is suitable for the determination of polyphenols in L. barbarum leaves, which provide polyphenols with suitable antioxidant activity.     

Analysis of Phenolic Compounds in Lager Beers of Different Origin: A Contribution to Potential Determination of the Authenticity of Czech Beer

Beer contains a wide range of polyphenolic compounds originating mainly from malt and hops. In this work newly modified on-line coupled HPLC–photodiode-array (PDA)–MS methods were used for analysis of characteristic phenolic compounds in several Czech lager beers, in comparison with some foreign lager beers. After optimization of column type, elution mode, and gradient steepness, chromatography was performed with a Restek Ultra Aqueous, C₁₈ (5 μm, 250 mm × 4.6 mm) column at 30 °C and gradient elution using an optimized linear gradient of aqueous acetonitrile acidified with 1% acetic acid, at a flow rate 0.4 mL min^?1. In total, 49 compounds were identified. Eleven individual compounds, predominantly malt phenolics (gallic acid, (?)-catechin, epicatechin, ferulic acid, chlorogenic acid, morin, rutin, quercetin, caempherol, naringenin, and luteolin) were quantified by use of two detection techniques: MS with electrospray ionization and UV detection. Compared with foreign beers, Czech beers contained higher levels of most of the phenolic compounds; specific distributions of individual compounds were also observed. Experimental PDA results for individual polyphenols were evaluated statistically by modified cluster analysis. Because of very tight covariance of the data a new procedure was devised for correlation analysis. The set of beers analyzed can be divided into four clusters closely related to the origin of the and the technology used. It seems that some of the flavonoids have potential use in beer authenticity analysis.     

Rapid and sensitive analysis of three polyphenols in tobacco by CE using homemade C(4) D with a mini detection cell

A rapid, sensitive, and practical CE with C(4) D detection was developed for the analysis of three polyphenols (rutin, scopoletin, and chlorogenic acid) in tobacco samples. The constructed mini detection cell (12 mm × 10 mm × 10 mm) of C(4) D featured with small inner cell volume (～2 nL), smaller noise (<0.9 mV), repeatability, high strength and durableness. Three polyphenols were ultrasonically extracted with methanol-water (70:30, v/v) solution following SPE cleanup. The CE method was optimized with the running buffer of 150 mmol L(-1) 2-amino-2-methyl-1-propanol (pH 11.2), and the applied separation voltage of +20 kV over a capillary of 50 μm id × 375 μm od × 50 cm (38 cm to the C(4) D window, 41.5 cm to the UV detector window), which gave a baseline separation of three polyphenols within ca. 6 min. The method provided the limits of quantification (S/N = 10) at about 0.08-0.15 μg g(-1) for three polyphenols, whereas the overall recoveries ranged from 82% to 88%. The proposed method has been successfully applied to measure three polyphenols in the actual tobacco samples, and their contents were calculated and evaluated.     

Simultaneous LC Determination of Chlorogenic Acid and Hydrochlorthiazide in Zhenju Jiangya Tablets

A method has been developed to determine chlorogenic acid and hydrochlorothiazide simultaneously in Zhenju Jiangya tablets by HPLC using an isocratic mode and UV detection. A solution of acetonitrile-0.4% phosphoric acid (13:87, v/v) was used as mobile phase. Results showed that the two compounds were well separated. Chlorogenic acid and hydrochlorothiazide had good linearities in the range of 0.64～6.4 μg mL^?1 and 0.08～0.16 mg mL^?1, respectively with average recoveries of 96.5～99.3 and 97.7～101.0%.     

Electrochemical reduction and quantification of menadione in sodium dodecyl sulfate micellar media

Menadione cyclic voltammograms show a pair of redox steps on glassy carbon electrode in 0.1 M H₃PO₄ with potential separation of 343 mV. Cationic, nonionic, and anionic surfactants micellar media significantly decrease the menadione peak potential separation. Statistically significant increase of menadione reduction current (3- and 4.4-fold) has been observed in Triton X 100 and sodium dodecyl sulfate (SDS) micellar media, respectively. Electrochemical reduction of menadione in 9 mM SDS micellar media is reversible diffusion-controlled one-electron process corresponding to formation of relatively stable semiquinone anion radical. The linear dynamic ranges of menadione determination are 7–560 and 600–2,550 μM with the limits of detection and quantification of 1.66 and 5.53 μM, respectively. The current concentration sensitivity is (8.6?±?0.2)?×?10³ μA μM^?1. The voltammetric method for the determination of menadione in pharmaceutical “Aekol” based on preliminary extraction with ethanol has been developed.     

Simultaneous Determination of Dopamine and Uric Acid by Electrocatalytic Oxidation on a Carbon Paste Electrode Using Pyrogallol Red as a Mediator

A sensitive and selective electrochemical method for the simultaneous determination of dopamine (DA) and uric acid (UA) was developed using a pyrogallol red modified carbon paste electrode. Under the optimized conditions, the peak current was linearly dependent on 1.0–700.0 μmol L^?1 DA and 50.0–1000.0 μmol L^?1 UA. The detection limits for DA and UA were 0.78 μmol L^?1 and 35 μmol L^?1, respectively. Finally, this method was also examined for the determination of DA and uric acid in real samples such as drugs and urine.     

Development and Validation of an SPE-LC Method for the Simultaneous Determination of trans-Resveratrol and Selected Flavonoids in Wine

A simple LC method has been developed for the simultaneous determination of the flavonols rutin, myricetin, quercetin, kaempferol and the stilbene, trans-resveratrol, in wines. Sample clean-up was performed with polymeric Nexus ABS ELUT cartridges. The polyphenols were separated in less than 25 min using gradient elution and UV detection at 320 nm. Average recoveries of the analytes from spiked wine ranged from 82.2 to 117.6% and the detection limits, based on spiked extracted synthetic wine, ranged from 0.02 to 0.2 mgL^?1. The method was applied to the analysis of Greek wines.