Reversed‐Phase High‐Performance Liquid Chromatography with Electrochemical Detection of Anthocyanins

Abstract

Anthocyanins, flavonoid compounds present in grapes and wines, were determined by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) with electrochemical detection (RP‐HPLC‐ED). The method developed consists of RP‐HPLC gradient elution with voltammetric detection using a glassy carbon electrode after separation in an Inertsil ODS‐3V analytical column. Good peak resolution was obtained following direct injection of a 50 µL sample of anthocyanins in a mobile phase of pH 2.20. The results show that six different anthocyanins: cyanidin‐3‐O‐glucoside chloride (kuromanin chloride), cyanidin‐3,5‐di‐O‐glucoside chloride (cyanin chloride), malvidin‐3‐O‐glucoside chloride (oenin chloride), malvidin‐3,5‐di‐O‐glucoside chloride (malvin chloride), delphinidin‐3‐O‐glucoside chloride (myrtillin chloride), and peonidine‐3‐O‐glucoside chloride, all with antioxidant properties, can be separated in a single run by direct injection of solution. The limit of detection (LOD) for these compounds was lower than 0.3 µM. The method can also be applied to the analysis of these compounds in red wines and in skins and pulp extracts of red grapes, since all these antioxidants are electroactive.     

Rapid quantitative analysis of individual anthocyanin content based on high‐performance liquid chromatography with diode array detection with the pH differential method

A new quantitative technique for the simultaneous quantification of the individual anthocyanins based on the pH differential method and high‐performance liquid chromatography with diode array detection is proposed in this paper. The six individual anthocyanins (cyanidin 3‐glucoside, cyanidin 3‐rutinoside, petunidin 3‐glucoside, petunidin 3‐rutinoside, and malvidin 3‐rutinoside) from mulberry (Morus rubra) and Liriope platyphylla were used for demonstration and validation. The elution of anthocyanins was performed using a C₁₈ column with stepwise gradient elution and individual anthocyanins were identified by high‐performance liquid chromatography with tandem mass spectrometry. Based on the pH differential method, the high‐performance liquid chromatography peak areas of maximum and reference absorption wavelengths of anthocyanin extracts were conducted to quantify individual anthocyanins. The calibration curves for these anthocyanins were linear within the range of 10–5500 mg/L. The correlation coefficients (r²) all exceeded 0.9972, and the limits of detection were in the range of 1–4 mg/L at a signal‐to‐noise ratio ≥5 for these anthocyanins. The proposed quantitative analysis was reproducible with good accuracy of all individual anthocyanins ranging from 96.3 to 104.2% and relative recoveries were in the range 98.4–103.2%. The proposed technique is performed without anthocyanin standards and is a simple, rapid, accurate, and economical method to determine individual anthocyanin contents.     

Quantitation of anthocyanins in elderberry fruit extracts and nutraceutical formulations with paper spray ionization mass spectrometry

The ability to rapidly identify and quantitate, over a wide range of concentrations, anthocyanins in food and therapeutic products is important to ensuring their presence at medicinally significant levels. Sensitive, yet mild, analysis conditions are required given their susceptibility to degradation and transformation. Paper spray ionization has been used to detect and quantify the levels of anthocyanin levels in extracts of fresh and dried elderberries, and elderberry stems, as well as 3 commercially available nutraceutical formulations. The component cyanidin glucosides, including cyanidin‐3‐sambubioside, cyanidin‐3‐glucoside, cyanidin‐3,5‐diglucoside, cyanidin‐3‐sambubioside‐5‐glucoside, and the aglycone cyanidin, were readily detected in a range of sources. Quantitation was achieved by establishing a calibration plot from dilutions of a stock solution of cyanidin‐3,5‐diglucoside containing malvidin‐3,5‐diglucoside as an internal standard at a fixed concentration. The same standard was used to quantify the anthocyanin content in the fruit and nutraceutical formulations. Wide 5‐fold variations in anthocyanin concentration were detected in the nutraceutical formulations from different suppliers ranging from 1050 to 5430 mg/100 g. These concentrations compared with 500 to 2370 mg/100 g measured in the dried stems and fruit, respectively.     

The Anthocyanins,Oenin and Callistephin,Protect RPE Cells Against Oxidative Stress

The retinal pigment epithelium (RPE) is a highly metabolic layer of postmitotic cells lining Bruch's membrane in the retina. While these cells contain endogenous photosensitizers that mediate blue light‐induced damage, it has also been shown that blue light exposure damages mitochondrial DNA in RPE cells resulting in mitochondrial dysfunction and unregulated generation of reactive oxygen species (ROS). As RPE cells are postmitotic, it is imperative to decrease oxidative stress to these cells and preserve function. Dietary plant‐derived antioxidants such as anthocyanins offer a simple and accessible solution for decreasing oxidative stress. The anthocyanins malvidin‐3‐O‐glucoside (oenin) and pelargonidin‐3‐O‐glucoside (callistephin) were tested for their ability and efficacy in decreasing ROS generation and preserving mitochondrial redox activity in blue light‐irradiated ARPE‐19 cells. A significant decrease in intracellular ROS with concurrent increase in mitochondrial redox activity was observed for tested concentrations of oenin, while callistephin was beneficial to stressed cells at higher concentrations. These findings suggest anthocyanins are effective antioxidants in blue light‐stressed RPE cells in vitro. Additionally, oxidation products of these anthocyanins were examined using LC/MS and findings suggest the possibility of multiple oxidation sites for these compounds.     

Specific Poly-phenolic Compounds in Cell Culture of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Gamay Fréaux

Cell cultures established from plants represent an attractive alternative to whole plants for effective production of bioactive secondary metabolites. Cell culture from Vitis vinifera L. cv. Gamay Fréaux accumulated high amounts of hydroxycinnamic acid derivatives and anthocyanins. Two new compounds were identified: 3-O-glucosylresveratrol, a stilbene derivative, abundant in cell suspension culture, and a hydroxyphenol, 4-(3,5-dihydroxyphenyl)-phenol, abundant in callus culture. The major anthocyanin monoglucosides present in cell suspension culture were cyanidin 3-O-glucoside and peonidin 3-O-glucoside, and the major cinnamoyl derivatives were cyanidin 3-O-p-coumaryl glucoside and peonidin 3-O-p-coumaryl glucoside. Three minor anthocyanin compounds were found in V. vinifera cell culture: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and delphinidin 3-O-p-coumaryl glucoside. Anthocyanin levels of cell suspension cultures increased significantly—about eight fold—after 4-day cultivation in new medium. Salicylic acid at a concentration of 50 μM did not enhance anthocyanin accumulation in cell suspension culture, and similar levels of jasmonic acid significantly reduced the anthocyanin content.     

Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips,stems, roots and leaves

The flavonoids and anthocyanins in Chinese sugarcane (Saccharum sinensis Roxb.) tips, stems, roots and leaves were separately analyzed by HPLC‐UV diode array detector, respectively. The results indicated that the content of flavonoids in sugarcane leaves was considerably high in comparison with previous reports in Brazil sugarcane (S. officinarum L.) leaves. Moreover, the content of flavonoids in sugarcane tips and roots was also high in comparison with sugarcane stems. For another, the content of anthocyanins in sugarcane roots was higher than that in other parts of the sugarcane, such as leaves, tips and stems. In addition, two anthocyanins, named petunidin 3‐O‐(6″‐succinyl)‐rhamnoside and cyanidin‐3‐O‐glucoside, were first identified from S. sinensis by HPLC‐UV diode array detector and HPLC‐MS/MS.     

Characterization and quantification of polyphenols in Amazon grape (Pourouma cecropiifolia Martius)

The phenolic profile of Amazon grape fruit (Pourouma cecropiifolia Martius) was investigated by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). For this purpose, suitable extraction and liquid chromatographic methods were developed. Anthocyanins, flavonols and chlorogenic acids were found mainly in the peel. Besides the main anthocyanins, i.e. delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-(6"-malonyl)glucoside, several minor anthocyanins were identified in the peel. Among these, cyanidin 3,5-diglucoside, delphinidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3-(3"-malonyl)glucoside, malvidin 3-glucoside, pelargonidin 3-glucoside, peonidin 3-glucoside and petunidin 3-glucoside were characterized on the basis of their fragmentation patterns in MS/MS experiments. The total anthocyanin content in the peel was 420.26±3.07 mg kg(-1) fresh weight. The pulp contained mainly 5-O-caffeoylquinic acid (210.39±3.43 mg kg(-1) fresh weight). Rutin was the predominant flavonol found in Amazon grape (peel 155.45 ± 2.06 mg kg(-1) fresh weight and pulp 2.64±1.21 mg kg(-1) fresh weight). Total polyphenols content was higher in the peel than in the pulp.     

Analysis and tentative structure elucidation of new anthocyanins in fruit peel of Vitis coignetiae Pulliat (meoru) using LC‐MS/MS: Contribution to the overall antioxidant activity

The skin of Vitis coignetiae Pulliat (meoru) grown wild in the Republic of Korea was analyzed for anthocyanins via HPLC coupled to ESI‐MS/MS in positive ion mode. Chromatographic separation was conducted via RP HPLC using a C₁₈ column, with a 50‐min gradient from 0 to 80% methanol in water containing 0.5% formic acid. A total of 18 anthocyanins were identified. Among them, nine compounds were newly determined by comparing the retention time (t_R) and mass fragmentation patterns with those of the previously reported anthocyanins for other grape varieties: malvidin hexose, peonidin 3‐galactoside, malvidin 3‐galactoside, cyanidin, petunidin, petunidin 3‐(6″‐coumaroyl)‐5‐diglucoside, peonidin, malvidin, and malvidin 3‐(6″‐coumaroyl)‐5‐diglucoside. The antioxidant activity of the V. coignetiae Pulliat anthocyanins was determined via 2,2‐diphenyl‐2‐picrylhydrazyl radical scavenging and 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation assays in a range of concentration from 25 to 500 mg/L. The capacity increased with concentration. The IC₅₀ values, defined as the concentration of sample required to scavenge 50% of free radicals, were calculated as follows: 189.63±11.31 mg/L and 141.29±6.70 mg/L for 2,2‐diphenyl‐2‐picrylhydrazyl and 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation, respectively. The antioxidant activity of the V. coignetiae Pulliat anthocyanins is substantially higher than that of ascorbic acid and is similar to the effects of the extracts obtained from other grape varieties.     

Rapid separation of cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside from crude mulberry extract using high‐performance countercurrent chromatography and establishment of a volumetric scale‐up process

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside, using high‐performance countercurrent chromatography, and the establishment of a volumetric scale‐up process from semi‐preparative to preparative‐scale. To optimize the separation parameters, biphasic solvent systems composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi‐preparative‐scale high‐performance countercurrent chromatography. The optimized semi‐preparative‐scale high‐performance countercurrent chromatography parameters (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200–1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative‐scale (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0–10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside. The separation of mulberry anthocyanins using semi‐preparative high‐performance countercurrent chromatography and its volumetric scale‐up to preparative‐scale was addressed for the first time in this report.     

Characterization and quantification of anthocyanins in selected artichoke (Cynara scolymus L.) cultivars by HPLC–DAD–ESI–MS n

The anthocyanin pattern of artichoke heads (Cynara scolymus L.) has been investigated by high-performance liquid chromatography–electrospray ionization mass spectrometry. For this purpose a suitable extraction and liquid chromatographic method was developed. Besides the main anthocyanins—cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3,5-malonyldiglucoside, cyanidin 3-(3′′-malonyl)glucoside, and cyanidin 3-(6′′-malonyl)glucoside—several minor compounds were identified. Among these, two peonidin derivatives and one delphinidin derivative were characterized on the basis of their fragmentation patterns. To the best of our knowledge this is the first report on anthocyanins in artichoke heads consisting of aglycones other than those of cyanidin. Quantification of individual compounds was performed by external calibration. Cyanidin 3-(6′′-malonyl)glucoside was found to be the major anthocyanin in all the samples analyzed. Total anthocyanin content ranged from 8.4 to 1,705.4 mg kg⁻¹ dry mass.      

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

Optimisation of pulsed electric fields extraction of anthocyanin from Beibinghong Vitis Amurensis Rupr

Beibinghong Vitis amurensis Rupr has wide plantation area, high productivity and rich anthocyanin. Common hot-extraction has poor deficiency and destroys anthocyanin severely. For Beibinghong V. amurensis Rupr as materials, response surface-optimised electric fields were used, the structure of Beibinghong was observed by SEM, antioxidant activity was measured by DPPH, ABTS and reducing force, the component of anthocyanin was analyzed by HPLC-MS. We found the content of total anthocyanin extracted by pulsed electric fields was 166.65 ± 3.88 mg/100 g.FW. Total anthocyanin from Beibinghong had high antioxidant activity, also contained multiple steady anthocyanin of delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside, delphinidin-3-O-(6-O-acetyl) glucoside and delphinidin-3-O-(6-O-p-coumaroyl) glucoside et al. In conclusion, the optimised pulsed electric fields method can quickly and efficiently extract several kinds of anthocyanins from V. amurensis Rupr. This study promoted the intensive processing of V. amurensis Rupr and widened the practical application of pulsed electric field technology.     

Characterization of phenolic compounds in green and red oak‐leaf lettuce cultivars by UHPLC‐DAD‐ESI‐QToF/MS using MSE scan mode

Lettuce (Lactuca sativa ) is one of the most popular leafy vegetables in the world and constitutes a major dietary source of phenolic compounds with health‐promoting properties. In particular, the demand for green and red oak‐leaf lettuces has considerably increased in the last years but few data on their polyphenol composition are available. Moreover, the usage of analytical edge technology can provide new structural information and allow the identification of unknown polyphenols. In the present study, the phenolic profiles of green and red oak‐leaf lettuce cultivars were exhaustively characterized by ultrahigh‐performance liquid chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization (ESI), and quadrupole time‐of‐flight mass spectrometry (QToF/MS), using the MS^E instrument acquisition mode for recording simultaneously exact masses of precursor and fragment ions. One hundred fifteen phenolic compounds were identified in the acidified hydromethanolic extract of freeze‐dried lettuce leaves. Forty‐eight of these compounds were tentatively identified for the first time in lettuce, and only 20 of them have been previously reported in oak‐leaf lettuce cultivars in literature. Both oak‐leaf lettuce cultivars presented similar phenolic composition, except for apigenin‐glucuronide and dihydroxybenzoic acid, only detected in the green cultivar; and for luteolin‐hydroxymalonylhexoside, an apigenin conjugate with molecular formula C₄₀H₅₄O₁₉ (monoisotopic MW = 838.3259 u ), cyanidin‐3‐O ‐glucoside, cyanidin‐3‐O ‐(3″‐O ‐malonyl)glucoside, cyanidin‐3‐O ‐(6″‐O ‐malonyl)glucoside, and cyanidin‐3‐O ‐(6″‐O ‐acetyl)glucoside, only found in the red cultivar. The UHPLC‐DAD‐ESI‐QToF/MS^E approach demonstrated to be a useful tool for the characterization of phenolic compounds in complex plant matrices.     

In‐time and in‐space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy

This paper presents a comprehensive analytical methodology, based on ‘in‐time’ and ‘in‐space’ tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan‐3‐ols and nine flavonols were determined by means of hyphenated high‐performance liquid chromatography – multi‐stage MS (HPLC‐MSⁿ) analyses (MSⁿ up to MS⁴), among which quercetin‐3‐O‐rutinoside‐7‐O‐glucoside, kaempferol‐3‐O‐rutinoside‐7‐O‐glucoside, quercetin‐3‐O‐galactosyl‐rhamnoside and quercetin‐3‐O‐coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC‐MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin‐3‐O‐rutinoside and cyanidin‐3‐O‐glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin‐3‐O‐rutinoside and kaempferol‐3‐O‐rutinoside were also found. Because flavonoids have been ascribed as potential health‐promoting compounds, gathered findings provide new insight into the knowledge of the quali‐quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry. Copyright © 2014 John Wiley & Sons, Ltd.     

Hexanuclear Zn(II) and Mononuclear Cu(II) Complexes containing imino phenol ligands: Exploitation of their Catalytic and Biological Perspectives

A unique hexanuclear zinc(II) ( 1 ) and two mononuclear copper(II) ( 2 and 3 ) complexes anchored with imino phenol ligand HL ¹ and HL ² were synthesized with good yield and purity (where HL ¹  = 4‐tert‐butyl‐2,6‐bis((mesitylimino)methylphenol and HL ²   =  5‐tert‐butyl‐2‐hydroxy‐3‐((mesitylimino)methyl)benzaldehyde). These complexes were characterized by utilizing various spectroscopic protocols like NMR, FTIR, UV as well as ESI‐Mass spectrometry, elemental analysis and single crystal X‐ray diffraction studies. Their potential to bind calf thymus DNA (CT‐DNA) was tested utilizing different techniques such as UV–visible and fluorescence spectroscopy. The experiment implies that they interact with CT‐DNA via non‐intercalative mode with moderate capabilities (K_b ~ 10⁴ M^?1). On the other hand, these complexes have high capabilities to quench the fluorescence of bovine serum albumin (BSA) following the static pathway. In addition, they are active catalysts for the oxidation reaction of 3,5‐di‐tert‐butylcatechol (3,5‐DTBC) to 3,5‐di‐tert‐butylquinone (3,5‐DTBQ) under aerobic condition. From the recorded EPR signals of all complexes, it has been concluded that the oxidation reaction proceeds via ligand oriented radical pathway instead of metal based redox participation. Kinetic studies using 1 – 3 indicate that it follows Michaelis–Menten type of equation with moderate to high turnover number (k_cat). Apart from these aspects, complexes 1 – 3 were screened for their cytotoxic behavior towards HeLa cells (human cervical carcinoma) and found quite active with comparable IC₅₀ values to cisplatin.     

Large‐scale isolation of high‐purity anthocyanin monomers from mulberry fruits by combined chromatographic techniques

Anthocyanins have attracted attention over the past several decades because of their beneficial health effects. In this research, a strategy combining column chromatography and high‐speed countercurrent chromatography was developed for the separation of high‐purity anthocyanin monomers from mulberry fruits. After purification using Amberlite XAD‐7HP column with 80% ethanol (0.1% HCl), a fraction of anthocyanins mixtures with a purity of 68.6% was obtained. High‐speed countercurrent chromatography with a biphasic solvent system of n‐butanol/methyl tert‐butyl ether/acetonitrile/water/trifluoroacetic acid (30:10:10:50:0.05, v/v) was used to separate the anthocyanin monomers. Three monomers of delphinidin‐3‐O‐ rutinoside, cyanidin‐3‐O‐ rutinoside, and cyanidin‐3‐O‐ glucoside were obtained, and identified by ¹H and ¹³C NMR spectroscopy and high‐performance liquid chromatography with electrospray ionization‐mass spectrometry. The method developed in this work can be used to conduct large‐scale separations of anthocyanin monomers from mulberry fruits and other plants.     

Purification and antioxidant capacity analysis of anthocyanin glucoside cinnamic ester isomers from Solanum nigrum fruits

In a recent study, anthocyanins, which have a strong free radical‐scavenging activity, were examined for their potential to effectively prevent cancer. However, clinical trials are limited by the purity of the anthocyanin. Multiple methods are used to extract and purify anthocyanins. Based on previous work on Solanum nigrum, which is a widely distributed plant, in this study, DM130 macroporous resin, Sephadex LH20, and a C18 column were used to separate cis–trans anthocyanin isomers. These anthocyanins constitute the majority of total S. nigrum anthocyanins. The results showed that this “DM130‐LH20‐C18 system” can be used to obtain a cinnamic acid‐derived cis–trans anthocyanin, petunidin‐3‐(p‐coumaroyl)‐rutinoside‐5‐glucoside, with a purity of 98.5%, for effective quantitation. In order to determine the antioxidant ability of the petunidin‐3‐(p‐coumaroyl)‐rutinoside‐5‐glucoside cis–trans isomers, three ordinary methods were adopted. The maximum antioxidant ability of the cis–trans anthocyanin was dozens of times higher than that of vitamin C.     

Reactivity of substituted copper(II) salicylates with tert‐butylperoxyl radical: Structure–reactivity relationships

Absolute rate constants (k_eff) for the chemical reactions of Cu(II)₂(3,5‐di‐iso‐propylsalicylate)₄(H₂O)₃, Cu(II)₂(3,5‐di‐tert‐butylsalicylate)₄, Cu(II)₂(3,5‐di‐tert‐butylsalicylate)₄(H₂O)₄, Cu(II)₂(3,5‐dimethylsalicylate)₄(H₂O)₃, Cu(II)₂(3‐ethylsalicylate)₄(H₂O), Cu(II)₂(3‐phenylsalicylate)₄, and Cu(II)(3,5‐di‐iso‐propylsalicylate)₂(pyridine)₂ with tert‐butylperoxyl radical were determined using kinetic electron paramagnetic resonance measurements in 10% toluene in the hexane medium at temperatures ranging from ?63°C to 2°C. These antioxidant (AO) chelates were ranked by their reactivity as follows: 2,6‐di‐tert‐butyl‐4‐methylphenol ? Cu(II)₂(3,5‐di‐tert‐butylsalicylate)₄ ? Cu(II)₂(3‐phenylsalicylate)₄ > Cu(II)₂(3,5‐di‐iso‐propylsalicylate)₄(H₂O)₃ ? Cu(II)₂(3,5‐di‐tert‐butylsalicylate)₄(H₂O)₄ ? Cu(II)₂(3,5‐dimethylsalicylate)₄(H₂O)₃ > Cu(II)₂(3‐ethylsalicylate)₄(H₂O) ? Cu(II)(3,5‐di‐iso‐propylsalicylate)₂(pyridine)₂ at 20°C. Differential pulse voltammetry was used to determine redox behavior of these chelates in CH₂Cl₂. Two types of salicylic OH groups were detected in these Cu(II) salicylates, characterized by the presence or absence of AO reactivity. One of them was coordinate covalently bonded to Cu(II) via the oxygen atoms of the salicylic OH groups, displaying oxidation peak potentials in the range from +650 to 970 mV versus Ag/Ag⁺. The second type was intramolecularly hydrogen bonded to carboxylate oxygens, with an oxidation peak potential in the range from +1100 to 1200 mV versus Ag/Ag⁺. It was concluded that non–hydrogen‐bonded salicylic OH groups are responsible for the antiperoxyl radical reactivity of these chelates, while neither Cu(II) nor salicylate ligands displayed reactivity with peroxyl radical. It has been established in this research that axially bonded electron pair donors such as pyridine and water decrease H‐donating reactivity of Cu(II) salicylates by promoting the formation of intramolecular hydrogen bonding between the salicylic OH hydrogen atoms and carboxylate oxygen atoms in the salicylic ligands. Dependences of log k_eff at 20°C and the anodic oxidation potential (E_pa) for the salicylic OH group on the difference between symmetric and asymmetric stretching frequencies of carboxylate groups (in Fourier transform infrared spectra) for the substituted Cu(II) salicylates were determined. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 42: 56–67, 2010     

Fast analysis of isobaric grape anthocyanins by Chip‐liquid chromatography/mass spectrometry

In a previous work, direct‐infusion electrospray ionization ion trap tandem mass spectrometry (ESI‐IT‐MS/MS) was applied to the study of anthocyanins in extracts from the skins of Clinton grapes, a non‐Vitis vinifera red grape variety qualitatively and quantitatively rich in anthocyanins. A good characterization of anthocyaninins was obtained, but it was impossible to differentiate some compounds with the same nominal mass but with different elemental composition. In this work, the capabilities of quadrupole time‐of‐flight mass spectrometry (QTOF‐MS) coupled with Chip‐liquid chromatography (LC‐Chip) were applied to the study of Clinton anthocyanins and this method provided the complete sample anthocyanin fingerprint in less than 5 min. Multi‐stage mass spectrometry (MSⁿ; n >2) was not necessary to identify isobaric compounds, nor were deuterium‐exchange experiments necessary to distinguish between compounds containing the same aglycone. The fast separation bypasses the problem of petunidin‐3‐O‐(6‐O‐acetyl)monoglucoside and delphinidin‐3,5‐O‐diglucoside quantification, present in the direct‐infusion ESI‐ITMS approach, due to overlapping with matrix interferences. Copyright © 2009 John Wiley & Sons, Ltd.     

Flavonol Glycosides from the Leaves of Embelia Keniensis

Five new flavonol glycosides characterized as syringetin 3‐O‐α‐rhamnoside‐7‐O‐β‐glucoside, syringetin 3‐O‐α‐rhamnoside‐7,4′‐di‐O‐β‐glucoside, quercetin‐7‐O‐β‐galactosyl (1→3)‐β‐galactoside, myricetin 3‐O‐α‐rhamnosyl (1→4)‐β‐galactoside and myricetin 3‐O‐β‐glucosyl (1→2)‐β‐glucoside‐7‐O‐β‐glucosyl‐(1→4)‐α‐rhamnoside have been isolated from a methanolic extract of Embelia keniensis leaves. Known flavonols isolated from the same extract included myricetin, quercetin, kaempferol, myricetin 3‐O‐α‐rhamnoside, myricetin 3‐O‐β‐glucoside, quercetin 3‐O‐α‐rhamnoside, quercetin 3‐O‐β‐glucoside, quercetin 3‐O‐β‐xyloside, isorhamnetin 3‐O‐α‐rhamnoside and myricetin 3‐O‐rutinoside. Their structures were established from extensive spectroscopic and chemical studies and by comparison with authentic samples.