Kinetic comparisons of anthocyanin reactivities towards 2,2'-azobis(2-amidinopropane) (AAPH) radicals, hydrogen peroxide and tert-buthylhydroperoxide by capillary zone electrophoresis

Twelve major anthocyanins identified in bilberry extracts were studied in vitro using capillary zone electrophoresis (CZE) for their reactions towards 2,2'-azobis(2-amidinopropane) (AAPH) radicals, hydrogen peroxides (H(2)O(2)) and tert-buthylhydroperoxides (t-BuOOH). Reactivity towards AAPH radicals was primarily determined by the aglycon structure, not by the type of sugar moiety. Delphinidins carrying three-hydroxyl groups on the B ring were most reactive followed by cyanidins, with two-hydroxyl groups. Further, methylation of the hydroxyl groups reduced reactivity towards AAPH radicals. However, reactivity of anthocyanins towards H(2)O(2) was not significantly affected by aglycon structure or by the type of sugar moiety; there being no marked difference in reaction rates among the anthocyanins. Reactivity towards t-BuOOH was essentially the same as towards H(2)O(2), although the reaction rate was several times smaller. Also, the reaction rate of anthocyanin towards peroxide was relatively high compared to that of (+)-catechin (approximately 30 times larger) measured as a reference antioxidant, whereas the reactivities of anthocyanins and (+)-catechin towards AAPH radicals were similar.     

Complete assignment of bilberry (Vaccinium myrtillus L.) anthocyanins separated by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) mobilities of fifteen anthocyanins in bilberry extract were completely characterized. Four minor anthocyanins in bilberry extract (malvidin 3-O-alpha-L-arabinopyranoside (Mv 3-ara), peonidin 3-O-beta-D-galactopyranoside (Pn 3-gal), peonidin 3-O-alpha-L-arabinopyranoside (Pn 3-ara), and petunidin 3-O-alpha-L-arabinopyranoside (Pt 3-ara)) that remained unidentified in our previous CZE study were isolated from the bilberry extract, and the chemical structures were assigned by NMR and MS. Their CZE mobilities were then precisely examined together with those of other major anthocyanins in the extract. When the CZE mobilities of the fifteen anthocyanins assigned here were plotted against their molecular weight/numbers of free phenolic group, it was found that separation of anthocyanins by CZE is primarily determined by the type of conjugated sugar present, and secondly by the aglycon structure.     

Structural dependence of HPLC separation pattern of anthocyanins from Bilberry (Vaccinium myrtillus L.)

An HPLC method using isocratic elution was established for the analysis of fifteen anthocyanins contained in bilberry (Vaccinium myrtillus L.). Separation was attained by using an aqueous solution of 20% methanol containing 0.5% TFA as the mobile phase with a flow rate of 2 ml/min. The detection limit was 0.3 pmol for delphinidin 3-O-beta-D-glucopyranoside, which is a major anthocyanin in bilberry extract. The reproducibility was 0.19-3.85% (S.E.M) for peak area and 0.64-0.77% (S.E.M) for relative mobility normalized by the elution position of the solvent peak. When the relative elution volumes of each anthocyanins were correlated to their corresponding anthocyanin structures, a characteristic pattern was observed. From this pattern, the structures of unknown anthocyanins could be predicted from their elution times. Therefore, the present method is useful for the study of anthocyanins from various biological sources.     

Simultaneous comparison of relative reactivities of twelve major anthocyanins in bilberry towards reactive nitrogen species

The reactivities of twelve major anthocyanins identified in bilberry extracts towards nitric oxide (NO.) and peroxynitrite (ONOO-) were studied in vitro using capillary zone electrophoresis (CZE). The reactivities of the anthocyanins towards NO. were slightly weak compared with that of (+)-catechin as a reference antioxidant under anaerobic conditions except delphinidin glycosides (Dp3glys). The reactivities of other anthocyanins were not significantly affected by either the aglycon structure or the type of sugar moiety. Under aerobic conditions, all anthocyanins and catechin showed significant enhancement of the reactivity, indicating that they reacted with other reactive species secondarily generated from NO. . Dp3glys showed rather extraordinally high reactivity towards ONOO- compared to other anthocyanins which showed approximately two times low reactivity than catechin when compared with IC50. Structural divergence in the reactivity was also small for all these anthocyanins.     

牡丹花色苷的热稳定性和降解动力学

研究了pH值和温度对牡丹花色苷热稳定性的影响。动力学数据分析表明,牡丹花色苷的热降解符合一级反应动力学模型。在70～90℃条件下,当pH值为2.6、3.0、3.6、4.0和4.6时,牡丹花色苷的半衰期分别为27.0～7.9、32.1～8.5、27.9～8.9、35.9～9.4和27.3～10.7h。提高温度,花色苷降解反应速率增大;降解反应速率常数与温度的关系附合Arrhenius公式,反应活化能为48.4～69.4kJ/mol。牡丹花色苷单体的降解速率依次为矢车菊-3-O-二葡萄糖苷芍药-3-O-葡萄糖苷矢车菊-3,5-O-二葡萄糖苷芍药-3,5-O-二葡萄糖苷。牡丹花色苷降解生成褐色物质,褐变指数随加热时间的延长、加热温度的升高、pH值的增大而增大。     

Analysis of anthocyanins in red onion using capillary electrophoresis-time of flight-mass spectrometry

For the first time, a capillary electrophoresis-time of flight-mass spectrometry analysis method for detecting anthocyanins in red onion was developed. The analysis method included the use of silica capillaries coated with poly-LA 313 (polycationic amine-containing polymer) and an MS-compatible volatile background electrolyte (BGE). The method was environmentally friendly and sensitive; and its rapidness combined with an acidic BGE helped in preventing anthocyanin degradation. By using high-resolution TOF-MS with pre-run tuning of masses, low mass errors were achieved in the determination of conjugated anthocyanins in red onion, and a simultaneous up-front fragmentation provided confirmation of the aglycon backbone for their secure identification. Most anthocyanins (at least seven out of ten) known in red onion from the literature were found, as well as one new for this matrix.     

CZE separation of strawberry anthocyanins with acidic buffer and comparison with HPLC

Anthocyanins, the major colourants of strawberries, are polar pigments that are positively charged at low pH. Herein, we have assessed a new analytical method for the separation of anthocyanins using CZE. Acidic buffer solutions (pH <2) were employed in order to maintain pigments in the cation flavylium form and achieve high molar absorptivity at 510 nm. These spectral properties enabled us to identify strawberry anthocyanins in a preliminary stage by detection in the visible range, although the method was optimised at 280 nm to obtain the best S/N. The effects of buffer composition highlighted the necessity of adding an organic modifier to the running buffer to obtain a suitable separation. The electrophoretic method permitted the separation of the three main anthocyanins of strawberry extracts, namely pelargonidin 3-glucoside (Pg-glu), pelargonidin 3-rutinoside and cyanidin 3-glucoside. The electrophoretic results, expressed as retention time and separation efficiency of the major anthocyanin (Pg-glu), were compared to those achieved in HPLC, the analytical technique traditionally used for the investigation of anthocyanins in vegetable matrix. The content of Pg-glu in strawberries (cv. Camarosa), calculated with HPCE and HPLC methods, resulted respectively in 11.41 mg/L and 11.37 mg/L.     

Hydrolysis of 3,4-diphenyl-1,2,5-thiadiazole-1,1-dioxide. Acid and base catalysis

We have performed a product and kinetic study of the hydrolysis of 3,4-diphenyl-1,2,5-thiadiazole-1,1-dioxide in aqueous solution. Benzil and sulfamide are the only products of hydrolysis and are formed in equimolar yields. The kinetic results indicate that a first order law is followed up to 90% conversion. The observed rate constant is independent of substrate concentration. Ionic strength and buffer concentration do not affect the rate constant, but the reaction is acid-base catalyzed. The rate-pH profile has been determined and a mechanism that fits the experimental data satisfactorily is proposed. Corresponding rate constants and equilibrium constants for the protonation of the substrate are reported.     

Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring

A systematic method for anthocyanin identification using tandems mass spectrometry (MS/MS) coupled to high-performance liquid chromatography (HPLC) with photo-diode array detection (PDA) was developed. Scan for the precursor ions of commonly found anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, and peonidin) using LC/MS/MS on a triple quadrupole instrument allows for the specific determination of each category of anthocyanins. Further characterization of each anthocyanin was performed using MS/MS product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM). The method was demonstrated for analysis of anthocyanins in black raspberries, red raspberries, highbush blueberries, and grapes (Vitis vinifera). Previous reported anthocyanins in black raspberries and red raspberries are confirmed and characterized. Common-neutral-loss analysis allows for the distinction of anthocyanin glucosides or galactoside and arabinosides in highbush blueberries. Separation and identification of anthocyanin glucosides and galactosides were achieved by LC/MS/MS using SRM. Anthocyanin isomers such as cyanidin sophoroside and 3,5-diglucoside were differentiated by their fragmentation pattern during product-ion analysis. Fifteen anthocyanins (all possible combinations of five anthocyanidins and three sugars) were characterized in highbush blueberries. Pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were detected and characterized for the first time in grapes. The present approach allows mass spectrometry to be used as a highly selective detector for rapid identification and characterization of anthocyanins and can be used as a sensitive procedure for screening anthocyanins in fruits and vegetables.     

Stability of anthocyanins extracted from grape skins

Summary The extractability and stability of anthocyanins from the skins ofVitis vinifera were determined at different pH values. Anthocyanins were extracted using acetone, partitioned with chloroform and pre-purified by solid-phase extraction (SPE). They were analysed by RP-HPLC, and the kinetic parameters of decomposition were calculated. The total monomeric anthocyanin content was determined by spectrophotometry. Anthocianins were well separated by RP-HPLC. The efficiency of extraction depended strongly on the pH of the extracting agent and on the character of the pigment to be extracted. The amount of anthocyanins decreased with increasing duration of storage, more so at elevated temperatures. Presented at Balaton Symposium '01 on High-Performance Separation Methods Siófok, Hungary, September 2–4, 2001.     

HPCE analysis of hydrolysing morphine derivatives. Quantitation of decomposition rate and mobility

Summary High performance capillary electrophoretic conditions were optimised on the basis of protonation constants and hydrolysis rate constants (if relevant) of 6 opiate compounds. Protonation constants were determined by pH-potentiometry, the progress of hydrolysis was followed by capillary electrophoresis, hydrolysis rate constants of ester-type compounds were elucidated by kinetic studies of the time- and pH-dependence of the decomposition. Using these physico-chemical parameters, the analysis circumstances were designed to keep thein situ hydrolysis rate negligible for every compound, which has not been the case in reported previous HPCE determinations of acetylated derivatives. Our calculated charge-related mobility differences and experimental CZE findings justified those earlier statements that capillary zone electrophoresis is insufficient to separate these compounds. The method development for diacetylmorphine, 3-acetylmorphine, 6-acetylmorphine, morphine, acetylcodeine and codeine resulted in the use of a micellar elecktrokinetic system operating at pH=8.0, applying 50 mM sodium dodecyl sulfate micelle-forming agent and 7.5% acetonitrile additive in the background electrolyte.     

Constituent analysis and quality control of anthocyanin constituents of dried Lycium ruthenicum Murray fruits by HPLC–MS and HPLC–DAD

In recent years, many investigations on the anthocyanins of the fresh Lycium ruthenicum Murray fruits have been reported; while few studies about dried fruits have been published. In this study, chemical profile of dried fruits was illustrated by a high-performance liquid chromatography–tandem mass spectrometry method, which provided evidence for the certain identification of the main anthocyanins. Among these compounds, nine of them were selected as marker compounds for the semiquantitative evaluation, using a simple and reliable method by high-performance liquid chromatography–photodiode array detection (HPLC–DAD), with the combination of chromatographic fingerprint analysis. Separation was achieved on a C18 ODS 80TS QA analytical column with linear gradient elution of acetonitrile and 10% formic acid and 0.1% trifluoroacetic acid (TFA) aqueous solution. Our results showed that the contents of anthocyanins of dried L. ruthenicum Murray from different origins were different. We also inferred the anthocyanin compositions of dried L. ruthenicum Murray through analyzing the UV spectrum, retention time, elution order, and MS data. Finally, eight kinds of anthocyanin compositions were identified and different from the anthocyanins in fresh L. ruthenicum Murray. In a word, this study may provide experimental data in further development and utilization of L. ruthenicum Murray.     

Kinetics and mechanism of the (-)-sparteine-mediated deprotonation of (E)-N-Boc-N-(p-methoxyphenyl)-3-cyclohexylallylamine.

The (-)-sparteine-mediated asymmetric lithiation-substitution of (E)-N-Boc-N-(p-methoxyphenyl)-3-cyclohexylallylamine ((E)-5) to afford gamma-substituted enantiomerically enriched products 6 is reported. The solution structure for the lithiated intermediate 8.1 in these reactions was determined by heteronuclear NMR to be a configurationally stable, alpha-lithio, eta(1)-coordinated monomer. This intermediate is proposed to exist as two rotamers that are rapidly equilibrating on the NMR time scale; competitive electrophilic substitution of each conformation results in the formation of Z or E products. Kinetic measurements of the lithiation by in situ infrared spectroscopy provide pseudo-first-order rate constants for reactions with a variety of concentrations of amine, (-)-sparteine, and n-BuLi. The reaction is first order in amine and zero order in 1:1 base--ligand complex. When the concentration of n-BuLi is varied independently of (-)-sparteine concentration, the reaction rate exhibits an inverse dependence on n-BuLi concentration. The deuterium isotope effect for the reaction was determined to be 86 at -75 degrees C, a result consistent with C--H bond breaking in the rate-determining step and indicative of tunneling. A reaction pathway involving a prelithiation complex is supported by kinetic simulations.     

Capillary electrophoresis-electrospray ionization-mass spectrometry method to determine the phenolic fraction of extra-virgin olive oil

We describe the first analytical method involving SPE and CZE coupled to ESI-IT MS (CZE-ESI-MS) used to identify and characterize phenolic compounds in olive oil samples. The SPE, CZE and ESI-MS parameters were optimized in order to maximize the number of phenolic compounds detected and the sensitivity of their determination. To this end we have devised a detailed method to find the best conditions for CE separation and the detection by MS of the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil (VOO). Electrophoretic separation was carried out using an aqueous CE buffer system consisting of 60 mM NH(4)OAc at pH 9.5 with 5% of 2-propanol, a sheath liquid containing 2-propanol/water 60:40 v/v and 0.1% v/v triethylamine. This method offers to the analyst the chance to study important phenolic compounds such as phenolic alcohols (tyrosol (TY), hydroxytyrosol (HYTY) and 2-(4-hydroxyphenyl)ethyl acetate), lignans ((+)-pinoresinol and (+)-1-acetoxypinoresinol), complex phenols (ligstroside aglycon (Lig Agl), oleuropein aglycon, their respective decarboxylated derivatives and several isomeric forms of these (dialdehydic form of oleuropein aglycon, dialdehydic form of ligstroside aglycon, dialdehydic form of decarboxymethyl elenolic acid linked to HYTY, dialdehydic form of decarboxymethyl elenolic acid linked to TY) and 10-hydroxy-oleuropein aglycon) and one other phenolic compound (elenolic acid) in extra-VOO by using a simple SPE before CE-ESI-MS analysis.     

FTIR analysis of hydrolysis in aliphatic polyesters

Infrared (IR) spectroscopy was adopted to study the hydrolytic degradation of films of aliphatic polyesters in alkaline environment. A measurable increase with the time of immersion of the absorbance of the peak centered at about 1570 cm⁻¹ was observed. Analysis of the IR spectra showed that the integrated peak area in that region can be used to quantify changes in the concentration of degradation products and thus to provide indications regarding the kinetic constant of the hydrolysis reaction. It was found that the hydrolysis of ester bonds proceeds linearly with time, and this result suggests that the controlling mechanism is the chemical reaction rather than water diffusion. The results also show that degradation rate increases with increasing polydispersity.     

Aerial oxidation kinetics of a phenolic porphyrin in acid solution

The rate of aerial oxidation of the title porphyrin ( 1 ) in dichloromethane (DCM), acidified with trifluoro-acetic acid (TFA), was found to be first-order in 1 and second-order in TFA at low TFA concentrations, and second-order in both 1 and TFA at higher TFA concentrations. At the lower TFA concentrations, aerial oxidation gives the two-electron oxidised product 2 , while at the higher TFA concentrations, a porphyrin pication radical 3 is formed. These results are rationalised by a kinetic scheme requiring, (i), preequilibrium involving transient formation of a porphyrin tetracation 4 , (ii), two-electron oxidation of 4 to a diprotonated oxidised porphyrin 5 , (iii) reaction of 5 with solvent water and (iv), conproportionation of 5 with 1 to generate 3 .     

Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine

A capillary zone electrophoresis (CZE) method is proposed for the quantitative determination of anthocyanins in wine as an alternative to high-performance liquid chromatography. The CZE separation was carried out using a 46 cm (effective length)×75 μm I.D. fused-silica capillary at 10 °C and a 50 mM sodium tetraborate buffer at pH 8.4 with 15% of methanol as modifier. A voltage of 25 kV and a hydrodynamic injection of 300 mbar s were used. The electropherograms were recorded at 599 nm. It was found that SO₂ (antibacterial and antioxidant agent added to wine during its production) increased the absorbance of anthocyanins at 599 nm in a basic medium. Therefore, a concentration of 250 mg/l of SO₂ was added to the samples and the calibration solution before the analysis in order to avoid errors by this matrix effect. The analytical response was linear (R=0.998) between 10 and 700 μg/ml of malvidin-3-O-glucoside. The limit of detection and the reproducibility (as a relative standard deviation, n=11) were 1 μg/ml and 1.5%, respectively. Finally, the CZE method was validated by the analysis of synthetic wine samples (errors less than 8%) and by the comparison of the results obtained in the analysis of different monovarietal wines by CZE with those obtained by the standard HPLC method. In this comparison, a good correlation (R=0.998) with a slope of 1.005±0.044 and an intercept of −0.752±6.690 was obtained for malvidin-3-O-glucoside.     

氮杂穴状大环钴(Ⅱ)配合物的合成、晶体结构及其催化性能

以一个氮杂穴状大环配体为自由配体,与硝酸钴经配合反应合成了一个新的穴状单核钴配合物[CoL(NO_3)]·(NO_3)·8H_2O(1),其晶体结构经X-射线单晶衍射表征。采用紫外-可见分光光度法对1催化p-硝基苯酚醋酸酯(NA)的水解动力学性质进行了研究。结果表明:催化水解速率对底物(NA)及配合物浓度均呈一级反应,催化水解速率受酸碱平衡影响。     

Identification of dimeric anthocyanins and new oligomeric pigments in red wine by means of HPLC-DAD-ESI/MSn

High-pressure liquid chromatography-diode array detector-electrospray ionisation/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)) analyses carried out in red wine fractions revealed the existence of dimeric anthocyanins (A-A(+)), previously detected in grape skin, and allowed the detection and identification, for the first time, of other derived oligomers. The structure of these compounds was characterised according to their MS(n)(n = 1-4) fragmentation patterns. The newly detected oligomers consisted of a flavanol, (epi)catechin or (epi)gallocatechin, linked through its C(4) position to the nucleophilic positions of the upper unit of a dimeric anthocyanin (F-A-A(+)). All the compounds contained malvidin as one of the anthocyanin subunits, whereas the other anthocyanin moiety could be either delphinidin, cyanidin, petunidin, peonidin or malvidin. With the fractionation method employed, the dimeric anthocyanins eluted in the same fractions as the monomeric anthocyanins. However, the new F-A-A(+) oligomers were found in the same fractions as F-A(+) dimers, which might indicate a structural similarity between both types of compounds. ESI/MS(n) analyses, coupled or not to HPLC, showed to be a useful and powerful tool for detecting and identifying these oligomers in wines, which usually elute from reversed-phase columns as humps and/or are overlapped by the peaks of other compounds. The detection of these oligomeric pigments in wine has provided more information about the complex pigments responsible for the elevation of the base line observed in the chromatograms of wines and has also revealed that oligomeric anthocyanins can take part in the reactions usually undergone by monomeric anthocyanins.     

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.