Caroline Mauve, Jean Bleton, Camille Bathellier, Caroline Lelarge-Trouverie, Florence Guérard, Jaleh Ghashghaie, Alain Tchapla and Guillaume Tcherkez* Plateforme Métabolisme Métabolome, IFR87 La Plante et son Environnement, Institut de Biotechnologie des Plantes, Université Paris-Sud XI, 91405 Orsay cedex, France LETIAM, Institut Universitaire de Technologie, BP127, Université Paris-Sud XI, 91405 Orsay cedex, France Ecologie, Systematique & Evolution, Université Paris-Sud XI, UMR 8079, 91405 Orsay cedex, France

We investigated the mechanisms involved in the retention of various peptides on a stationary phase embedded with a quaternary ammonium group (BS C23), by high-performance liquid chromatography. This was compared with peptide retention on a conventional reversed-phase C18 (RP C18) column under isocratic conditions, to understand better the various mechanisms involved. Chromatographic characterization of the two stationary phases with “model” compounds showed that BS C23 is less hydrophobic than RP C18 and induces electrostatic interaction (attraction or repulsion) with ionized compounds. If reversed-phase partitioning was the predominant retention phenomenon, for both stationary phases, the retention mechanisms in BS C23 provided different selectivity to that of RP C18. Electrostatic attraction or repulsion was clearly observed between peptides and the permanent positively charged group embedded in BS C23 depending on the pH. For most of the peptides, a weak anion-exchange mechanism was observed on the quaternary ammonium-embedded stationary phase if mobile phases at neutral pH and low ionic strengths were employed.

COMPOSITION OF GLYCERIDIC FRACTION OF 4 CUCURBITACEAE SEED SPECIES FROM CONGO BRAZZAVILLE WAS STUDIED IN ORDER TO EVALUATE INTRA ET INTER SPECIFIC VARIABILITY. THIS IS A PRE-CONDITION TO ANY SELECTION WORK BECAUSE SEEDS WITH LOWER VARIABILITY LEAD TO CONSTANT QUALITY OILS. THE PRESENT STUDY SHOWS THAT 2 SPECIES (CUCURBITA MOSCHATA (CM) AND CITRULLUS LANATUS (CL)) HAVE LOWEST INTRA SPECIFIC VARIABILITY, CONSIDERING ALL OF FATTY ACIDs, FA (CV < 20%). THEIR SEEDS LEAD TO HOMOGENOUS POPULATION. IF ONE TAKES IN ACCOUNT LINOLEIC ACID (C18:2), THE MOST IMPORTANT ESSENTIAL FATTY ACID OF EDIBLE OILS, THIS VARIABILITY FALLS RESPECTIVELY TO 20% AND 10%; CONSEQUENTLY THE SEEDS OF THE 4 SPECIES (CUCURBITA MOSCHATA, CITRULLUS LANATUS (CL), CUCURBITA PEPO (CP) AND LAGENARIA SICERARIA (LS)) PRESENT THE SAME NUTRITIONAL INTEREST. INTER SPECIFIC VARIABILITY WHICH IS HIGHER THAN INTRA ONE, LEADS TO A GOOD SEPARATION OF THE SPECIES. MULTIVARIATE ANALYSIS (ANALYSIS IN PRINCIPAL COMPONENTS (ACP), ASCENDING HIERARCHICAL CLUSTERING (AHC) CONFIRMS THIS RESULTS AND HIGHLIGHTS THE HOMOGENEITY OF EACH SPECIES SEEDS AND THE GOOD DISCRIMINATION OF THE DIFFERENT SPECIES.

Fatty acids composition of various classes of lipids (neutral and polar lipids) was determined by gas chromatography (GC) and triacylglycerols (TAG) composition by high performance liquid chromatography (HPLC). The comparative study of fatty acids and triacylglycerols composition of the five cucurbitaceae species analyzed allow similarities within the species. The essence of the linoleic acid C 18:2 are concentrated in the TAG and the acids linoleic C 18:3 and palmitoleic C 16:1 can be used as facto r discriminating the studied species.

The purpose of this study was to find a simple and rapid method allowing the simultaneous quantification of some alcoholic fermentation inhibitors present in aqueous distillery effluent in order to evaluate its recycling properties. A capillary gas chromatography (CGC) method was tested for the quantification of both short chain fatty acids (acetic to hexanoic) and neutral compounds (butane 2,3-diol, 2-furaldehyde, phenyl-2-ethane1-ol). A polyvalent column coated with ®trifluoro-propyl-polysiloxane, allowing water injection, was tested and experiments were performed directly on untreated samples. During the development of the method, a deformation of acid peaks was observed; that could be explained by a secondary equilibrium, added to the chromatographic equilibrium. Although the acid peaks were deformed, calibration curves were produced and rigorously validated, proving that quantification is possible even when the best chromatographic conditions have not been achieved. Eventually, the method enabled the concentration of eight major fermentation inhibitors in distillery effluent to be measured.

HPLC analysis of dyes from ancient objects is often difficult because of the small quantity of colorant extracted; their identification, usually based on DAD, is limited to commercially available compounds. It was shown that the structural characterisation of unknown compounds could be achieved using HPLC–MS and could be finely tuned to enable both detection and characterisation. Optimisation was carried out using source parameter step-adjustment, mobile phase composition and post-column additive testing; dye characterisation trial was carried out using MS3 pattern study. The results clearly show advantages in comparison to DAD namely improved molecular identification and better selectivity. Unfortunately, as several compounds had the same molecular mass and MS3 fragmentation, their retention parameters were very useful for adequate structure recognition. The optimised conditions were successfully applied to the identification of components from a small sample of wool thread dyed with madder (Rubia tinctorum) and Our Lady's bedstraw (Galium verum).

Amongst the numerous base-deactivated ODS phases obtained by increasing the bonding density or/and by efficient endcapping treatments, some particular stationary phases have been developed, to limit the additional interactions of basic compounds with residual silanols, to work at extreme pH or with rich water mobile phases. Horizontal polymeric phases, sterically protected ones, hybrid silicas, propylene bridge, are particularly used for this purpose. Octadecyl chains with embedded polar groups and hydrophilic endcapping are also used in this goal. The properties of these phases were studied with a simple test consisting in the injection of carotenoid pigments in Subcritical Fluid Chromatography. The molecules used and the nature of the mobile phase allow the determination of hydrophobicity, polar site accessibility and type or/and bonding density of the stationary phases. Whatever the type of the phases, the particular stationary phases do not show any remarkable property, in comparison to other base-deactivated C18-bonded phases. On the other hand, embedded and polar-endcapped phases display a specific behaviour in regard of hydrophilic interactions, which are highlighted by the absence of water in the subcritical fluid. Additional properties of these phases are described, such as steric recognition and retention performances. As expected, polar-embedded phases are less retentive than classical ODS ones, but are sometimes able to provide greater steric recognition. On the other hand, the polar-endcapped phases display greater hydrophobicity than polar-embedded ones. From a simple classification diagram based on chromatographic properties, differences can be noticed between the polar-embedded groups (amide, carbamate, ether, sulfonamide) and between embedded and endcapped phases. Surprising behaviours are also noticed for some on the tested phases.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Strategies aimed at treating atherosclerosis by immunization protocols are emerging. Such protocols commonly use adjuvants as non-specific stimulators of immune responses. However, adjuvants are known to modify various disease processes. The aim of this study was to determine whether adjuvants alter the development of atherosclerosis. We performed immunization protocols in apolipoprotein E knockout mice (E degrees ) following chronic administration schedules commonly employed in experimental atherosclerosis. Our results point out a dramatic effect of several adjuvants on the development of atherosclerosis; three of the four adjuvants tested reduced lesion size. The Alum adjuvant, which is the adjuvant currently used in most vaccination protocols in humans, displayed a strong atheroprotective effect. Mechanisms accounting for atheroprotective effect of Freund's adjuvants included their capacity to increase both Th2 responses and anti-MDA-LDL IgM titers, and/or to impose atheroprotective lipoprotein profiles. The present study indicates that adjuvants have potent atheromodulating capabilities, and thus, implies that the choice of adjuvant is crucial in long-term immunization protocols in experimental atherosclerosis.

Six different olibanum samples with certified botanical origin were analyzed by headspace SPME-GC/MS in order to define their mono-, sesqui- and diterpenic composition, as pertinent criteria of identification. Boswellia carteri and Boswellia sacra olibanum have quite similar chemical composition, with isoincensole acetate as the main diterpenic biomarker. Although Boswellia serrata olibanum also exhibits this biomarker, the presence of methylchavicol, methyleugenol and an unidentified oxygenated sesquiterpene distinguishes B. serrata olibanum from the two other species. The characteristic chemical compounds of Boswellia papyrifera are the diterpenic biomarkers incensole and its oxide and acetate derivatives, n-octanol and n-octyl acetate. Boswellia frereana olibanum is devoid of diterpenes of the incensole family but contains a high amount of many dimers of alpha-phellandrene. The chemical composition of olibanum, which is demonstrated to be different for each Boswellia species allowed the determination of the taxonomic origin of frankincense samples purchased on various markets in East Africa, in the Near East and in Yemen. Moreover, terpenic fingerprints allowed the botanical origin of olibanum used in traditional incense mixtures to be identified. Furthermore, this study gave us the opportunity to assign a botanical origin to an archaeological frankincense sample.

A liquid chromatography method for the characterization of base deactivated columns was investigated in a collaborative study involving six laboratories. This work was carried out on two chromatographic supports (Xterra RP 18 and Symmetry Shield). Different cooling systems, namely water bath and air oven, were tested and it was shown that column thermoregulation did not significantly influence chromatographic data. In order to control the mobile phase composition, the latter was prepared by weight rather than volume. Thanks to the injection of a set of selected neutral compounds, extra-column effects were evaluated in each of the participating laboratories. The results showed that chromatographic supports tested in different laboratories and following the same test protocol could be effectively compared.

This paper describes a new test designed in subcritical fluid chromatography (SFC) to compare the commercial C18 stationary phase properties. This test provides, from a single analysis of carotenoid pigments, the absolute hydrophobicity, the silanol activity and the steric separation factor of the ODS stationary phases. Both the choice of the analytical conditions and the validation of the information obtained from the chromatographic measurements are detailed. Correlations of the carotenoid test results with results obtained from other tests (Tanaka, Engelhard, Sander and Wise) performed both in SFC and HPLC are discussed. Two separation factors, calculated from the retention of carotenoid pigments used as probe, allowed to draw a first classification diagram. Columns, which present identical chromatographic behaviors are located in the same area on this diagram. This location can be related to the stationary phase properties: endcapping treatments, bonding density, linkage functionality, specific area or silica pore diameter. From the first classification, eight groups of columns are distinguished. One group of polymer coated silica, three groups of polymeric octadecyl phases, depending on the pore size and the endcapping treatment, and four groups of monomeric stationary phases. An additional classification of the four monomeric groups allows the comparison of these stationary phases inside each group by using the total hydrophobicity. One hundred and twenty-nine columns were analysed by this simple and rapid test, which allows a comparison of columns with the aim of helping along their choice in HPLC.

Irvingia gabonensis est un arbre largement repandu en Afrique. Son aire de distribution geographique s'etend du Nigeria en Angola. Des nombreux travaux a caractere ethnobotanique soulignent l'importance et l'interet que les populations autochtones de ces regions accordent a son fruit et particulierement a son amande qui, a plus de 60%, contient une graisse alimentaire qui pourrait avoir des utilisations industrielles en margarinerie, en savonnerie et en cosmetique. Malheureusement il n'existe aucun travail systematique sur le plan chimique et technologique. Cet article contribue a combler cette lacune. L'analyse totale (proteines, lipides, cendres et elements mineraux) de l'amande est suivie d'une etude detaillee de l'extraction (dans differents solvants) et de la caracterisation (proprietes physico-chimiques et composition chimique) du beurre d'Irvingia. Cette etude permet de mieux entrevoir les valorisations possibles de cette ressource locale abondante dans le Golfe de Guinee, en Afrique du centre et de l'ouest.

The aim of this study was to use headspace solid phase microextraction (SPME) to reveal the presence of resin in archaeological samples, such as mummification balms, from ancient Egypt. Experiments were first performed with fresh resins of known origin. The SPME fibre readily extracted mono- and sesquiterpenes and, to a lesser extent, diterpenes. Using mass spectra and retention indices of constitutive compounds, qualitative analysis of the volatile fraction allowed us to differentiate resins or gum-resins such as myrrh, olibanum, galbanum, labdanum, mastic, and conifer resins. SPME was then successfully applied to archaeological samples from ancient Egypt in which the presence of resins was detected. Volatile components were desorbed and trapped according to the same SPME procedure as was applied to fresh resins, after a sample preparation consisting of a fine grinding.

Gas chromatography-mass spectrometry has been used to determine the nature of organic materials used in mummification balms. A comparative analysis of samples taken from Egyptian mummies is developed. The results are given in two parts. First, it is shown that the chemical composition of the balm is practically independent of the part of the mummy from which it is taken. This study was done on a Ptolemaic mummy (circa 100 BC from the Guimet Museum in Lyon). Fats, beeswax, and diterpenic resins were the main components: they were found everywhere. Castor oil was also very often detected (in half of the samples). This particular fat is present in the balm inside the thorax but not in the skull. Moreover it is shown that a vegetable tannin was employed. Components indicative of vegetable tannin input (gallic acid and inositols) were found in seven samples out of eighteen, particularly close to the body and on the canopic pack of the heart. Secondly, some conclusions from a comparative study of the composition of balms from mummies of various social levels as well as of different Egyptian periods are reported. It is shown that beeswax was used as from very early times (XVIIIth dynasty). The mixture of beeswax, fats, and diterpenoid resins would appear to be more recent. The balms of three mummies dating from more recent Egyptian periods (XIXth to XXVth dynasty) were analysed. No evidence of a resin, gum-resin, or plant gum could be found. Some mummies would appear to have been embalmed with fats or beeswax. Finally, the entrails canopic pack said to belong to Ramses II undoubtedly shows an embalming process with a triterpenic resin of the mastic type. The adopted analytical methodology enabled us to achieve simultaneous detection of four components of the balm of the Ptolemaic mummy. Analysis of the other five mummies revealed far less complex chemical compositions for the balms. This may be an indication of different embalming processes, although we should bear in mind the question of organic matter preservation through the ages.

Numerous relationships usually used in high-performance liquid chromatography (HPLC) for describing the retention on porous graphitic carbon (PGC) have been applied in subcritical fluid chromatography, with CO2-methanol mobile phases. As reported in HPLC, octanol-water partition coefficient failed to fit the retention, whereas satisfactory results were obtained with the sum of partial negative charges. A better fit was reached by using the solvation parameter model, allowing a better understanding of the interactions developed between the solute, the stationary and the mobile phases. Results show that the dominant contribution to retention was given by the polarizability (E) and the volume (V), while the hydrogen-bond basicity (B) was not selected in the retention model, whatever the methanol content. The increase in methanol percentage favours the retention decrease, mainly through the volume for hydrophobic compounds, and through the hydrogen-bond acidity for polar compounds.

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.

The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand-ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25-1.30 to 0.51 or increases from 1.25-1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve (b = 1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.