Maria Careri

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd2+) can result in cell death, but the molecular mechanisms of Cd2+ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl2 and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl2 treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by NG-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd2+-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd2+ cytotoxicity in plants.

Contribution of Dynamic Headspace GC-MS Analysis of Aroma Compounds to Authenticity Testing of Honey.

Abstract The volatile profiles of 43 authentic honey samples of different botanical and geographical origins were obtained by means of gas chromatography–mass spectrometry. A qualitative analysis of the volatile compounds identified was performed in order to assess the marker compounds (if/when existing) for both botanical and geographical origin. The results seem to indicate the existence of certain marker compounds for the floral origins assessed (e.g. acacia, chestnut, eucalyptus, heather, lavender, lime, rape, rosemary and sunflower). Also such compounds for two geographical origins (e.g. Denmark and England) seem to exist and possible marker compounds could also be found for the honeys from The Netherlands, Spain and Portugal.

Recent advances in the application of mass spectrometry in food-related analysis.

A review is presented on recent applications of mass spectrometry (MS)-based techniques for the analysis of compounds of food concern. Substances discussed are naturally occurring compounds in food products such as lipids, oligosaccharides, proteins, vitamins, flavonoids and related substances, phenolic compounds and aroma compounds. Among xenobiotics, applications of MS techniques for the analysis of pesticides, drug residues, toxins, amines and migrants from packaging are overviewed. Advances in the analysis of trace metals of nutritional and toxicological interest by MS with inductively coupled plasma (ICP) source are presented. The main features of mass spectrometry combined with separation instruments are discussed in food-related analysis. Examples of mass spectrometry and tandem MS (MS-MS) are provided. The development and application of matrix-assisted laser desorption ionization (MALDI) and electrospray (ESI) to the analysis of peptides and proteins in food is discussed. This survey will attempt to cover the state-of-the-art up from 1999 to 2001.

Overview of the applications of liquid chromatography–mass spectrometry interfacing systems in food analysis: naturally occurring substances in food

Abstract This paper reviews applications of different LC–MS techniques for the analysis of natural compounds in foods. Specific examples of substances discussed are lipids, oligosaccharides, vitamins, flavonoids and related substances, phenolic compounds, glucosinolates, and other miscellaneous naturally occurring compounds in food products. LC–MS is a powerful technique in food analysis and especially for analysis of complex mixtures, where additional analytical information is required to confirm positively the identity of the separated compounds or few separations are obtained. Among the interfacing systems used to couple LC with MS, the newly developed electrospray/ionspray mass spectrometric liquid interface offers undoubted advantages in terms of sensitivity and capability to analyze large, thermally labile and highly polar compounds; in addition, tandem MS techniques are useful for structural elucidation studies.

Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil

A narrow-bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 microg/ml and quantitation at concentrations of 0.52 and 0.54 microg/ml for sitosterol and stigmasterol, respectively. An excellent linearity was determined over two orders of concentration magnitude (r2 0.999-1.000) and verified by applying the Mandel fitting test (p>0.099) and the lack-of-fit test (p>0.057) performed at the 95% confidence level. A good intra-day precision ranging from 0.15 to 1.16% was calculated at two concentration levels (2 and 100 microg/ml). The inter-day reproducibility was verified on 3 different days by performing an homoscedasticity test and analysis of variance. A solid-phase extraction method was developed on silica cartridges for the isolation of phytosterols from soybean oil providing recovery values of 101+/-9 and 106+/-7% for sitosterol and stigmasterol, respectively. Good accuracy of the method was statistically demonstrated since no matrix effect was found for both the analytes. The developed method was applied to the quantitative assay of phytosterols in a soybean oil sample (61+/-5 mg/100 g of stigmasterol and 118+/-4 mg/100 g sitosterol). The HPLC-atmospheric pressure chemical ionization MS technique enabled the identification of stigmasterol, sitosterol and campesterol in the oil sample.

Spectrophotometric and coulometric detection in the high-performance liquid chromatography of flavonoids and optimization of sample treatment for the determination of quercetin in orange juice.

The capabilities of spectrophotometric and electrochemical detection techniques were investigated for the high-performance liquid chromatographic determination of flavonoids. Liquid chromatographic analyses were performed on eleven compounds belonging to three different classes of flavonoids: flavanone glycosides, flavone and flavonol aglycones. Separation of all compounds examined was carried out under reversed-phase conditions on a C18 narrow-bore column for UV detection, whereas for electrochemical detection, a C18 standard-bore column was used. UV analyses were carried out at 280 nm for flavanones and at 265 nm for flavones and flavonols, whereas controlled-potential coulometric measurements were performed using a porous graphite electrode. Analytical performances of the methods were compared in terms of linearity, limits of detection (LODs) and precision. Linearity over two orders of magnitude and LODs at low-ppm levels (0.06-1 mg/l) were demonstrated for all techniques considered. Instrumental precision in terms of relative standard deviation was found to be between 0 and 5% for the liquid chromatography (LC)-UV system and between 0.6 and 10% for the LC-electrochemical detection (ED) system. The methods developed were applied to the analysis of flavanones and flavonols in a real sample, such as an extract of orange juice. Even though quercetin glycoside is mostly present in orange juice as rutin, other different glycosides of this flavonol could be present; on this basis, the hydrolysis of all glycosides to aglycone allows one to obtain more accurate data on the flavonol concentration in orange juice. To avoid sample degradation and to increase extraction efficiency, quercetin hydrolysis was optimized using a central composite design to investigate the effects of acid concentration and hydrolysis time on extraction recovery.

Applications of liquid chromatography-mass spectrometry interfacing systems in food analysis : pesticide, drug and toxic substance residues

This paper reviews applications of different LC-MS techniques for the determination of xenobiotic substances in foods. Specific examples of contaminants discussed are pesticides, herbicides, insecticides and drugs; concerning toxic substances, mycotoxins, phycotoxins, cyanobacterial toxins, mutagenic and heterocyclic amines and beta-carbolines, arsenic, tin and inorganic halogen compounds, packaging materials and various epoxy resins are considered. Advantages and limitations are outlined for the different LC-MS interfacing systems (particle beam, thermospray, atmospheric pressure ionization with electrospray, ionspray and heated pneumatic nebulizer). The impact of developments in instrumental analysis on methodology and the limitations of the various LC-MS methods are discussed. Further, the coupling of LC with element-selective detection systems such as inductively coupled plasma mass spectrometry is discussed, with emphasis on speciation of trace toxic elements in foods.

Multiplexed Determination of Protein Biomarkers Using Metal-Tagged Antibodies and Size Exclusion Chromatography−Inductively Coupled Plasma Mass Spectrometry

A liquid-phase immunoassay was developed for the simultaneous determination of five cancer biomarker proteins: alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), ovarian tumor antigen (CA125/MUC16), and gastrointestinal tumor antigen (CA19-9). The method was based on the incubation of a serum (or tissue cytosol) with five antibodies, each labeled with a different lanthanide (Pr(3+), Eu(3+), Gd(3+), Ho(3+), and Tb(3+), respectively) followed by the specific determination of the immunocomplex formed by size exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICPMS). The sensitivity of the method was comparable with that attainable by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay with the advantages of multiplexed analysis capacity, virtually no sample preparation, and sample amount consumption, ca. 3 times lower than an ELISA test. The method was validated for the analysis of the proteins in human serum and proved to be able to discriminate ovary and uterus tumor tissue samples from those of healthy subjects.

Pentacopper(II) 12-metallacrown-4 complexes with α- and β-aminohydroxamic acids in aqueous solution: a reinvestigation

A reinvestigation of the equilibria of (S)-α-alaninehydroxamic acid (α-Alaha) and (R)-aspartic-β-hydroxamic acid (Asp-β-ha) with copper(II) was performed in aqueous solution in order to clarify some contradictory literature reports regarding the stoichiometry of the polynuclear complexes formed. β-Alaninehydroxamic acid (β-Alaha, HL), for which the formation of a planar 12-metallacrown-4, [Cu5L4H−4]2+, was already reported, was also re-examined for comparison. Among the different techniques used (potentiometry, absorption spectrophotometry, spectropolarimetry and electrospray ionization mass spectrometry), ES data allowed to define unambiguously that all these three ligands form the same pentanuclear species. Therefore it can be concluded that in aqueous solution the hydroxamates of both α- and β-amino acids form 12-metallacrown-4 complexes, and that the formers are less stable.

Validation of a liquid chromatography ionspray mass spectrometry method for the analysis of flavanones, flavones and flavonols.

The application of liquid chromatography/mass spectrometry (LC/MS) with a TurboIonspray (TIS) interface was investigated as a new method for the analysis of flavonoids. Eleven compounds belonging to three different classes of flavonoids were studied: eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin (flavanone glycosides), quercetin, kaempferol, galangin (flavonol aglycones), chrysin, apigenin (flavone aglycones). Chromatographic separations were performed under reversed-phase conditions using a C18 narrow-bore LC column; a mixture of an aqueous solution of formic acid (pH 2.4) and acetonitrile was used as the mobile phase. Isocratic elution was operated in the case of flavanones, whereas gradient elution was used for the simultaneous separation of flavones and flavonols. The adaptability of TIS to high flow applications allows the use of LC eluent flow rates at 200 µL/min without post-column splitting. Qualitative analysis was performed in negative-ion (NI) full-scan mode, whereas response linearity, detection limits and precision of the method were studied under NI selected ion monitoring (SIM) conditions. Characterization of isomers differing in the glycosylation was found to be possible on the basis of different mass spectra. Detection limits in the low-ng range (0.08-0.4 ng) were found, about twenty-fold lower than those reported previously. The method was applied to identify and determine the content of flavonoids in an orange juice sample. Copyright 1999 John Wiley & Sons, Ltd.