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.     

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.     

Anthocyanins HPLC-DAD and MS Characterization,Total Phenolics,and Antioxidant Activity of Some Berries Extracts

Extracts of indigenous wild blackberries, mulberries, bilberries, and blackthorns were analyzed for anthocyanin composition, anthocyanin content, total phenolics, and antioxidant capacity. Anthocyanins extraction with acidified methanol in ultrasonic condition (59 kHz, 60 min., 25°C) was carried out. The extracts were analyzed by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 apparatus equipped with photodiode array detector for qualitative characterization of the anthocyanins. The chromatograms revealed the presence of a large number of anthocyanins in fruits extracts: blackberries, 4 compounds; mulberries, 3 compounds; bilberries, 18 compounds; and blackthorns, 5 compounds. The most abundant anthocyanins were cyanidin-3-glucoside in blackberry, mulberry, and bilberry, and cyanidin-3-rutinoside in blackthorn extract. Structural information about anthocyanins was obtained by using a mass spectrometric method based on fully automated chip-nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT). Anthocyanin content was quantified by the pH differential method and total phenolics were determined by Folin-Ciocalteu method. A Jasco V 530 UV-VIS spectrophotometer was used for absorbance measurements. The free radical scavenging activity of the berries extracts was performed by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The reduction of DPPH was followed by a spectrophotometric method. Also, a correlation of the antioxidant capacities of the extracts with their anthocyanin content and total phenolics was attempted.     

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.     

Rapid screening for anthocyanins and anthocyanin dimers in crude grape extracts by high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry

A rapid and efficient method using high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry (HPLC-DAD–MS/MS) for fast screening large numbers of anthocyanins and anthocyanin dimers in different grape skin extracts, without further sample clean-up procedures, was developed. A good separation of most detected anthocyanins was achieved in a run time of 15 min. Identification of anthocyanin pigments required a combination of several information: UV–vis spectra, MS and MS/MS spectra, and elution pattern. Many compounds have been here detected for the first time and their structures tentatively elucidated.     

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.     

Comparison of Different Cartridges of Solid Phase Extraction for Determination of Polyphenols in Tobacco by UPLC/MS/MS and Multivariate Analysis

The comparison of solid phase extraction(SPE) for the preconcentration and isolation of polyphenols in tobacco samples was carried out by ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and multivariate analysis.Several adsorbing materials of SPE(C18,NH2,SAX and OASIS) were investigated.It was found that the C18 and OASIS cartridges can not only speed up the purification process,but also simplify the SPE operation.A UPLC/MS/MS was used for the determination of polyphenols ...     

General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography–electrospray ionization-ion trap-time of flight-mass spectrometry

A systematic approach for optimizing the extraction and identification of anthocyanins from blueberries was explored using HPLC-UV and HPLC–ESI-IT-TOF-MS. Sample homogenization effects, extraction solvent selection, type of acid, and amount used in extraction solvent were investigated. A mixture of methanol:water:trifluoroacetic acid (70:30:1, v/v/v) was found to be the best solvent system for blueberry anthocyanin extraction. Differences in total anthocyanin content due to commercial blueberry processing were explored as an application using the optimized extraction technique and HPLC-UV analysis. A methodical system for anthocyanin identification by HPLC–ESI-IT-TOF-MS without the use of standards was also reviewed and applied. Consideration was given to elution order by chromatographic separation with selective detection at 520 nm, high mass accuracy m/z values, tandem MS fragmentation, and previously published literature. Overall, 25 anthocyanins from a wild type highbush blueberry were identified and reported.     

High-resolution triacylglycerol mixture analysis using high-temperature gas chromatography / mass spectrometry with a polarizable stationary phase,negative ion chemical ionization,and mass-resolved chromatography

High-temperature gas chromatography/mass spectrometry (HT-GC/MS) has been employed to study the behavior of mixtures of triacylglycerol molecular species on a polarizable stationary phase (immobilized 65% phenyl methyl silicone). The use of negative ion chemical ionization (NICI) at an ion source block temperature of 300 °C overcomes problems with interpretation of electron ionization (EI) mass spectra produced during the HT-GC/MS. The NICI spectra of triacylglycerols produced under these conditions contain abundant [RCO₂]^?, [RCO₂ ? 18]^?, and [RCO₂ ? 19]^? ions, believed to be produced by nucleophilic gas-phase ammonolysis, that are used to identify the individual fatty acid moieties associated with peaks in triacylglycerol total ion chromatograms. The polarizable stationary phase produces significantly enhanced resolution of triacylglycerol molecular species compared to hightemperature stable apolar stationary phases, such as immobilized dimethyl polysiloxanes. The resolution of complex natural mixtures of triacylglycerols can be further improved by use of the Biller-Biemann enhancement technique to produce mass-resolved chromatograms. Investigation of the mass-resolved chromatograms provides important information with regard to the factors that affect elution orders of individual triacylglycerol molecular species. The analysis of mixtures of authentic triacylglycerols by HT-GC/MS via NICI provides data that relate to the analytical limits of the technique for the analysis of triacylglycerols that bear both saturated and polyunsaturated fatty acyl moieties.     

Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques

A combination of column chromatography on Toyopearl gel HW-40 (S) and polyamide resin has been developed for the preparative isolation and further determination of pyranoanthocyanins of oligomeric nature formed after reaction between anthocyanins and different flavanols in a complex wine matrix. Polyamide chromatography was found to be exceptionally useful to separate oligomeric pyanoanthocyanins from other classes of wine flavonoids and polymerized pigments into an advanced state of purity for further identification and quantification by HPLC-diode array detector coupled with electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS). Fractionation on Toyopearl gel chromatography allowed the separation of pyranoanthocyanins bearing the same flavanols (catechin, epicatechin and procyanidin dimers) but with different anthocyanin moieties (either acylated or non-acylated in the glucose residue) in order to allow further isolation of individual oligomeric pigments on C18 chromatography. A quantitative procedure for analyzing the major pyranoanthocyanin-flavanol derivatives in different aged wines is proposed for the first time. Results obtained showed good reproducibility and recovery regarding sample pretreatment and quantitative method for all analyzed oligomeric pyranoanthocyanins. The combination of these two chromatographic separations is likely to be applicable to the preparative isolation of other anthocyanin-derived pigments.     

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.     

High-Throughput Platform for Novel Reaction Discovery

Detecting the formation of new chemical bonds in high-throughput synthesis is limited by the efficiency and scalability of reaction product detection, as conventional methods for isolating product from reaction mixtures are time consuming and labor intensive. Here, we report a miniaturizable purification method that enables the rapid, high-throughput isolation of quaternary ammonium-tagged products from reaction mixtures with excellent purity using inexpensive equipment that easily can be set up in a typical organic chemistry laboratory. This novel purification technique enabled us to establish a high-throughput reaction discovery platform. We validated this platform in a screen of 1536 reactions, and one previously unreported transformation was identified.     

Anthocyanins as antimicrobial agents of natural plant origin

Anthocyanins are particularly abundant in different fruits, especially in berries. The beneficial effects of these compounds for human health have been known from at least the 16th century. Despite the great number of papers devoted to the different biological effects exerted by anthocyanins only a limited number of studies is focused on the antimicrobial activity of these compounds. Anthocyanin content of berry fruits varies from 7.5 mg/100 mg fresh fruit in redcurrant (Ribes rubum) up to 460 mg/100 g fresh fruit in chokeberry (Aronia melanocarpa). After consumption, anthocyanins are intensively metabolized, mainly in the intestines and liver. Glucorination, methylation and sulfation are the most typical metabolic reactions. Antimicrobial activity of crude extracts of plant phenolic compounds against human pathogens has been intensively studied to characterize and develop new healthy food ingredients as well as medical and pharmaceutical products. However, there is very little information available about the antimicrobial activity of the pure anthocyanins. In the last part of this review we present the collection of papers describing the anthocyanin profiles of different fruits (mainly berries) and the antimicrobial properties of the identified compounds. Generally, anthocyanins are active against different microbes, however Gram-positive bacteria usually are more susceptible to the anthocyanin action than Gram-negative ones. Mechanisms underlying anthocyanin activity include both membrane and intracellular interactions of these compounds. Antimicrobial activity of berries and other anthocyanin-containing fruits is likely to be caused by multiple mechanisms and synergies because they contain various compounds including anthocyanins, weak organic acids, phenolic acids, and their mixtures of different chemical forms. Therefore, the antimicrobial effect of chemically complex compounds has to be critically analyzed.     

Zirconia layer coated mesoporous silica microspheres as HILIC SPE materials for selective glycopeptide enrichment

Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides.     

Identification of microcystin-RR and [Dha7]microcystin-RR in commercial standards by electrospray ionization mass spectrometry

Three different commercial standards of microcystin-RR were assessed for purity by the liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) technique. Although the liquid chromatograms with photodiode array detector for each standard looked virtually identical, the analysis of corresponding mass spectra revealed that only one of them contained microcystin-RR per purity assay. The second standard was a mixture of microcystin-RR, and its demethyl variant identified as [Dha7]microcystin-RR, and the third one contained [Dha7]microcystin-RR only. We strongly recommend applying LC coupled with MS for purity assay of microcystin standards.     

Utilization of capillary electrophoresis/mass spectrometry (CE/MSn) for the study of anthocyanin dyes

Hyphenation of capillary electrophoresis with electrospray ionization mass spectrometry was utilized for the monitoring of anthocyanins in wine and wine musts. CE/MS was performed in two electrolytes: 1) an acidic one (chloroacetate-ammonium, pH 2) and 2) a basic one with high selectivity towards derivatives containing vicinal hydroxy groups (borate-ammonium, pH 9). The setup of MS was optimized and the fragmentation of common anthocyanins was studied in detail. Attention was also focused on the fragmentation of anthocyanidin skeleton. The anthocyanidins substituted with hydroxy groups fragment via a cascade of neutral losses of water and carbon monoxide. Fragmentation of anthocyanidins containing a methoxy group on their B-ring starts with the cleavage of methane and/or methyl radical. The optimized method was utilized for the monitoring of changes in anthocyanin profile in red wines as well as the process of release of anthocyanins to wine must.     

Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE

Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States.     

Preparation and Application of a Novel Silica-Supported Organic-Inorganic Hybrid Molecular Imprinting Polymer

Abstract

A novel estazolam-imprinted silica sorbent was prepared by the surface imprinting technique using 3-aminopropyltriethoxysilane (APTEOS) and phenyltrimethoxysilane (PTMOS) as functional monomers. The functional monomers are expected to form hydrogen bonds and π-π interactions with estazolam. The imprinted silica sorbent was characterized by Fourier transform infrared spectroscopy (FT-IR), element analysis, and scanning electron micrograph (SEM). Compared to C₁₈ solid phase extraction (SPE) and liquid-liquid extraction, molecular imprinting polymer (MIP) SPE was the most feasible method to extract estazolam from human plasma, and the recovery of estazolam was up to 98.7±1.2%.     

Purity assessment of commercially available crystalline deoxynivalenol

Deoxynivalenol (DON) obtained from 2 commercial sources was characterized, and its purity was determined. The structural identity of DON was confirmed by 1H and 13C-nuclear magnetic resonance (NMR) spectroscopy, gas chromatography with mass spectrometric (GC/MS) detection, and infrared/attenuated total reflectance (IR/ATR) spectroscopy. NMR spectra showed shifts that varied from previously published data. However, we established a complete, unambiguous assignment for all signals. Chromatograms obtained by GC/MS were almost identical for both investigated samples and confirmed the structure of DON. Likewise, IR/ATR spectra verified the identity of DON. The degree of purity was determined by liquid chromatography (LC) with a variable wavelength detector, LC/MS/MS, GC with electron-capture detection (GC-ECD), and ultraviolet (UV) spectrophotometry. The purity check using LC showed a single peak in both chromatograms. With LC/MS/MS measurements, we could detect small amounts of impurities in the crystalline DON from both sources. In data obtained by GC-ECD, no differences in purity were observed. The UV measurements showed an absorption maximum at 217 nm. The mean epsilon(m) of the extinction coefficients was calculated as 6727 (L/cm/mol) for DON (Sigma) and 6825 (L/cm/mol) for DON (Biopure). Finally, the purity of DON from the 2 commercial sources was calculated as >96 and >98%, respectively. Although the DON produced by both providers can be considered sufficiently pure for routine analysis of trichothecenes in food and feed, this work again demonstrated that the impurity of the solid mycotoxin constitutes the greatest contribution to the overall uncertainty of a mycotoxin calibrant.     

An information-based computational technique for estimation of chromatographic peak purity

Assessment of the purity of chromatographic peaks is an important step in developing and validating purification procedures for complex mixtures. While curve-fitting techniques can be useful for determining the retention times and relative concentrations of the components of a chromatographic peak, their utility is limited by the lack of unambiguous criteria for determining the number of such components. In this work, we present a computational technique for analyzing chromatograms to estimate the number of components, their retention times, and their relative concentrations. In contrast to Fourier-transform-based techniques, the technique we present does not require manual peak identification. It is based on curve-fitting and uses the Akaike information criterion to estimate the number of components. Application of the technique to chromatograms obtained from size-exclusion and reverse-phase chromatography of test mixtures indicates that it is useful for the characterization of complex mixtures.