Paola Dugo

COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY-MASS SPECTROMETRY: A REVIEW

Although comprehensive two-dimensional gas chromatography (GC x GC) has been on the scene for more than 15 years, it is still generally considered a relatively novel technique and is yet far from being fully established. The revolutionary aspect of GC x GC, with respect to classical multidimensional chromatography, is that the entire sample is subjected to two distinct analytical separations. The resulting enhanced separating capacity makes this approach a prime choice when GC analysts are challenged with highly complex mixtures. The combination of a third mass spectrometric dimension to a GC x GC system generates the most powerful analytical tool today for volatile and semi-volatile analytes. The present review is focused on the rather brief, but not scant, history of comprehensive two-dimensional GC-MS: the first experiments were carried out at the end of the 1990s and, since then, the methodology has been increasingly studied and applied. Almost all GC x GC-MS applications have been carried out by using either a time-of-flight or quadrupole mass analyzer; significant experiments relative to a variety of research fields, as well as advantages and disadvantages of the MS systems employed, are discussed. The principles, practical and theoretical aspects, and the most significant developments of GC x GC are also described.

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E2 (PGE2). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants. The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM). In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.

Release of Protein, Lipid, and Vitamin E from Almond Seeds during Digestion

The evaluation of the bioaccessibility of almond nutrients is incomplete. However, it may have implications for the prevention and management of obesity and cardiovascular disease. This study quantified the release of lipid, protein, and vitamin E from almonds during digestion and determined the role played by cell walls in the bioaccessibility of intracellular nutrients. Natural almonds (NA), blanched almonds (BA), finely ground almonds (FG), and defatted finely ground almonds (DG) were digested in vitro under simulated gastric and gastric followed by duodenal conditions. FG were the most digestible with 39, 45, and 44% of lipid, vitamin E, and protein released after duodenal digestion, respectively. Consistent with longer residence time in the gut, preliminary in vivo studies showed higher percentages of nutrient release, and microscopic examination of digested almond tissue demonstrated cell wall swelling. Bioaccessibility is improved by increased residence time in the gut and is regulated by almond cell walls.

LC-MS for the identification of oxygen heterocyclic compounds in citrus essential oils

The oxygen heterocyclic compounds (coumarins, psoralens and polymethoxylated flavones) present in the nonvolatile residue of the essential oils of Mandarin, Sweet Orange, Bitter Orange, Bergamot and Grapefruit were analysed with an HPLC/API/MS system equipped with an APcI probe in positive mode. The use of hyphenated techniques, such as LC/MS provides a great information about the content and nature of constituents of natural complex matrices, such as essential oils. In this work, MS spectra were recorded at different voltages, to obtain structural information in addition to molecular weight information. The different response of the compounds identified has been also evaluated. The method allowed the confirmation of the identification of the main components of the fraction, previously reported for the different oils. MS characteristics of coumarins, psoralens and polymethoxylated flavones with different substitution patterns were determined on the basis of the response obtained with the APcI interface. Interface parameters were optimised to obtain a contemporaneous response for all the three classes of components.

Detailed analysis and group-type separation of natural fats and oils using comprehensive two-dimensional gas chromatography

Comprehensive gas chromatography (GC x GC) is an adequate methodology for the separation and identification of very complex samples. It is based on the coupling of two capillary columns that each give a different but substantial contribution to the unprecedented resolving power of this technique. The 2D space chromatograms that derive from GC x GC analysis have great potential for identification. This is due to the fact that the contour plot positions, pinpointed by two retention time coordinates, give characteristic patterns for specific families of compounds that can be mathematically translated. This investigation concerned the application of this principle to fatty acid methyl esters that were grouped on an equal double bond number basis. The ester samples were derived from various lipids and all underwent bidimensional analysis on two sets of columns. Peak attribution was supported by mass spectra, linear retention indices and information reported in the literature.

Characterization of 12 Capsicum varieties by evaluation of their carotenoid profile and pungency determination.

In this research 12 different varieties of Capsicum cultivars belonging to three species (Capsicum chinense, Capsicum annuum, Capsicum frutescens) and of various colour, shape, and dimension have been characterised by their carotenoids and capsaicinoids content. The berries were cultivated in the region Emilia-Romagna, in Northern Italy. The native carotenoid composition was directly investigated by an HPLC-DAD-APCI-MS methodology, for the first time. In total, 52 carotenoids have been identified and considerable variation in carotenoid composition was observed among the various cultivars investigated. Among the cultivars with red colour, some Habanero, Naga morich and Sinpezon showed an high β-carotene content, whereas Serrano, Tabasco and Jalapeno showed an high capsanthin content and the absence of β-carotene. Habanero golden and Scotch Bonnet showed a high lutein, α-carotene and β-carotene amounts, and Habanero orange was rich in antheraxanthin, capsanthin and zeaxanthin. Cis-cryptocapsin was present in high amount in Habanero chocolate. The qualitative and quantitative determination of the capsaicinoids, alkaloids responsible for the pungency level, has also been estimated by a validated chromatographic procedure (HPLC-DAD) after a preliminary drying step and an opportune extraction procedure. Results have also been expressed in Scoville units. Dry matter and water activity have also been established on the fresh berries. The dried peppers of each variety were then submitted to the evaluation of the total nitrogen content, measured by a Dumas system, permitting to provide information on the protein content that was found to be in the range between 7 and 16%.

Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection.

The combined use of the state-of-the-art hybrid mass spectrometers together with high efficient liquid chromatography could surely be a useful tool for such a challenging task, as phospholipids (PLs) analysis. In this research, we used hydrophilic interaction liquid chromatography (150 mm×2.1 mm I.D., 2.7 μm d.p. partially porous column) to achieve the separation of major PLs classes in cows and donkeys milk samples. Solid-phase extraction (SPE) was performed in order to pre-concentrate minor PLs from non polar lipids (triacylglycerols) and the recovery for the extraction method was assayed on a milk sample, fortified with 5 μg/mL of SM pure standard, and analyzed in triplicate. A value of 89.99% was calculated, with a coefficient of variation (CV%) of 1.93. A 70-min long stepwise gradient of water/acetonitrile afforded baseline separation of PLs classes, at 50 μL/min flow rate. Accurate detection by an ion trap-time of flight (IT-TOF) mass spectrometer (in both positive and negative ionization mode) allowed to fully characterize the distinctive phospholipid profile and fatty acid composition of cows and donkeys milk, the latter being analyzed for the first time. Evaporative light scattering detection was further employed to attain the quantitative evaluation of major PLs classes identified, by the external calibration method using reference material solutions in the 5-200 μg/mL concentration range. Major difference between the two analyzed samples consisted in the total PLs amount, which in cows milk was determined as over 20-fold higher than the donkeys.

Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts

Stevia rebaudiana extracts and plant materials are increasingly used as natural sweeteners. Polyphenolic and stevioside compounds contained in S. rebaudiana extracts were separated by comprehensive LC. A polyamine column operated in normal phase mode was used for the first dimension separation (D1), and a UHPLC C18 column operated in reversed phase mode was used for the second dimension separation (D2). The sub-2 μm column (2.1 mm × 30 mm, maintained at 70°C) and the UHPLC pump employed for D2 elution allowed a separation/cycle time of 20 s, with a backpressure oscillating between 805 and 922 bar at 3.4 mL/min. The reduced D2 cycle time allowed 3-12 D2 samplings for each peak eluted by D1. Polyphenolic and stevioside compounds were identified by combining the information coming from the position of the compounds in the 2D plot and UV spectra with that of reference materials.

Mass spectrometry detection in comprehensive liquid chromatography: Basic concepts, instrumental aspects, applications and trends

The review, as can be deduced from the title, focuses on both theoretical and practical aspects of the use of mass spectrometry as a third, added dimension to a comprehensive LC (LC × LC) system, generating the most powerful analytical tool today for non-volatile analytes. The first part deals with the technical requirements for linkage of an LC × LC system to an MS one, including the choice of the mobile phase (buffer and salts), flow rate (splitting), type of ionization (interface); advantages and disadvantages of off-line and on-line methods are discussed, as well. A discussion of the various aspects of instrumentation is provided, both from a chromatographic and mass spectrometry standpoint, with particular emphasis directed to the choice of column sets, spatial resolution, mass resolving power, mass accuracy, and tandem-MS capabilities. The extent to which mass spectrometry may be of aid in unraveling column-outlet multicompound bands is highlighted, along with its effectiveness as a chromatographic detector of excellent sensitivity, universality yet with potential in terms of selectivity and amenability to quantitative analysis over a wide dynamic range. The following section of the review contains significant applications of comprehensive two-dimensional LC coupled to MS in different areas of research, with details on interfaces, column stationary phases, modulation and MS parameters. It is not the intention of the authors to provide a comprehensive description of the techniques, but merely to discuss only those aspects which are essential for successful applications of the LC-MS combination. The reader will be acquainted with the enormous potential of this hyphenated technique, and the factors and instrumental developments that have concurred to make it emerge to a central role in specialized fields, such as proteomics.