Marie-Claire Hennion

SOLID-PHASE EXTRACTION METHOD DEVELOPMENT, SORBENTS, AND COUPLING WITH LIQUID CHROMATOGRAPHY

The objective of this review is to provide updated information about the most important features of the new solid-phase extraction (SPE) materials, their interaction mode and their potential for modern SPE. First, the recent developments are given in formats, phases, automation, high throughput purpose and set-up of new types of procedures. Emphasis is then placed on the large choice of sorbents for trapping analytes over a wide range of polarities, such as highly cross-linked copolymers, functionalized copolymers, graphitized carbons or some specific n-alkylsilicas. The method development is given which is based on prediction from liquid chromatographic retention data or solvation parameters in order to determine the main parameters of any sequence (type and amount of sorbent, sample volume which can be applied without loss of recovery, composition and volume of the clean-up solution, composition and volume of the desorption solution). Obtaining extracts free from matrix interferences in a few steps--one step when possible--is now included in the development of SPE procedure. New selective phases such as mixed-mode and restricted access matrix sorbents or emerging phases such as immunosorbents or molecularly imprinted polymers are reviewed. Selectivity obtained by combining two sorbents is described with the use of ion-exchange or ion-pair sorbents. Special attention is given to complete automation of the SPE sequence with its on-line coupling with liquid chromatography followed by various detection modes. This represents a fast, modern and reliable approach to trace analysis. Many examples illustrate the various features of modern SPE which are discussed in this review. They have been selected in both biological and environmental areas.

Immuno-based sample preparation for trace analysis.

Immuno-based sample preparation techniques are based upon molecular recognition. Thanks to the high affinity and high selectivity of the antigen-antibody interaction, they have been shown to be a unique tool in the sampling area. Immuno-based sample preparation methods include the widely encountered immunoaffinity extraction sorbents, so-called immunosorbents, as well as membrane-baed or ultrafiltration techniques. This review describes the new developments and applications that have occurred in recent years with emphasis on (i) the antigen-antibody interactions, (ii) and their importance for the properties and use of immunosorbents, (iii) multiresidue extractions, (iv) the on-line coupling to chromatographic or electrophoretic separations, and (v) the high potential for improving MS detection. The recent use of artificial antibodies for sample pretreatment, so-called molecularly imprinted polymers, is also described.

Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review

Abstract Immunoassay techniques provide a simple, powerful and inexpensive method for pesticide analysis. However, the acceptance of immunoassays is dependent on the demonstration of quality and validity compared to more traditional techniques. In this review, primarily, the knowledge and the fundamentals of immunoassay methods are given in order to make good use of immunoassays, especially of ELISA tests. Special attention is given to a better understanding of the high selectivity and sensitivity which is attained for some immunoassays and not for others. It is also explained why some immunoassays are a quantitative method whereas others can only be used as a screening method. The cross-reactivity process, the effect of the sample matrix and the data interpretation are illustrated by numerous examples from the literature. Other formats, especially flow-injection immunoassays, dipstick immunoassay and liposome-amplified immunoassays are presented. Quality assurance and guidelines for validation and use are given.

Graphitized carbons for solid-phase extraction.

The objective of this review is to provide updated information about the most important features of graphitized carbonaceous sorbents used for solid-phase extraction (SPE) of organic compounds from liquid natural matrices or extracts. The surface characteristics of graphitized carbon blacks and porous graphitic carbons are described which are responsible for the various types interactions (hydrophobic, electronic and ion-exchange) with analytes. The method development is given which is based on the prediction from liquid chromatographic retention data obtained using porous graphitic carbon. Emphasis is placed on their capability for trapping very polar and water-soluble analytes from aqueous samples. Comparison is made between carbon-based SPE sorbents and other reversed-phase materials such as octadecyl silicas and highly cross-linked copolymers. Especially, the difficulty encountered for the desorption of some strongly retained analytes is explained by LC data and solutions are given for optimizing the composition and volume of the desorption solution. Many examples illustrate the various common features of graphitized carbons which are the extraction of very polar analytes and multiresidue extractions. Some applications are specific to graphitized carbon black due to the presence of surface functional groups. They include the extraction of anionic compounds such as benzene and naphthalene sulfonates or acidic pesticides. Other applications are specific to porous graphitic carbon due to its flat and homogeneous surface. One example is the trace extraction of coplanar polychlorinated biphenyls (PCBs), dibenzo-p-dioxins and dibenzofurans from other PCB congeners.

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.

Influence of the number and length of alkyl chains on the chromatographic properteis of hyrdrocarbonaceous bonded phases

Abstract Non-polar bonded stationary phases have been prepared by reaction of organochlorosilanes with silica. The influence of the surface coverage on chromatograpic properties was studied. The capacity ratio and selectivity do not increase continuously with increasing surface coverage and reach maximum at a surface coverage of ca. of 2 μmol·m−2 for polar and non-polar solutes. The influence of the length of the bonded alkyl chains at constant surface coverage was studied. The capacity ratio and selectivity increase with increasing length of bonded bristles. A compromise between surface coverage, alkyl chain length and mobile phase composition is discussed.

Effect of the various parameters governing solid-phase microextraction for the trace-determination of pesticides in water

The parameters governing solid-phase microextraction (SPME) are investigated, with emphasis on the determination of the partition coefficients, K, and their use to predict the selection of a fibre, depending on the respective characteristics of the fibres and the analytes. Film thickness and stability of the compounds can interfere with the determination of K values. The time profile curves were determined for twelve pesticides having a wide range of water solubilities and polarities and using four fibres (polydimethylsiloxane, polydimethylsiloxane-divinylbenzene, Carbowax-divinylbenzene and polyacrylate). Although the affinity order was different for the four coatings, no correlation was found between the respective characteristics of the fibres and solutes. The two fibres containing divinylbenzene were shown to have the highest affinities and the polydimethylsiloxane had the lowest affinity. The polyacrylate fibre which is the more polar commercial fibre did not provide the highest affinities for the more polar and water-soluble analytes. The important parameters for quantitative analysis have been evaluated. The calibration curves were similar when one analyte of interest was present on its own in a drinking water sample, or when eleven other pesticides were present at the same concentration or when much higher concentrations of other analytes were present in the sample. Linearity was obtained over a wide range of concentrations in drinking water samples. Detection limits are in agreement with European regulatory levels in drinking water for most of the analytes using solid-phase microextraction-gas chromatography-nitrogen-phosphorus detection (SPME-GC-NPD). In contaminated surface water samples, the chromatograms are relatively clean and most of the compounds can be detected at levels lower than 0.5 microgram/l.

Immunoaffinity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices

Immunoaffinity solid-phase extraction (SPE) sorbents, so-called immunosorbents (ISs), are based upon molecular recognition using antibodies. Thanks to the high affinity and high selectivity of the antigen-antibody interaction, they allow a high degree of molecular selectivity and have shown to be a unique tool in the sample preparation area these last few years. Extraction and clean-up of complex biological and environmental aqueous samples are achieved in the same step and from large volumes when required. Their application to extracts from solid matrixes is solvent-free and more simple than any other clean-up procedure. Single analytes can be targeted, but since an antibody can also bind one or more analytes having structure similar to the one used for its preparation, ISs have been developed for targeting a single analyte and its metabolites. The cross-reactivity was also exploited for developing ISs that could selectively extract a whole class of structurally related compounds. This review describes the current technology used for the synthesis of the ISs, their properties and their field of application. The different parameters governing the antigen-antibody interactions and the solid-phase extraction process are discussed. Emphasis is given to the optimisation of the SPE sequence, especially to the desorption and regeneration steps. The importance of the capacity and its relationship with the analytes recovery and breakthrough volumes is highlighted for class-specific ISs. Multi-class-selective ISs are also presented. Validation studies are reviewed using various certified reference materials. Relevant examples, involving combination with chromatography in both off-line and on-line mode, illustrate the high selectivity provided in various complex matrixes. Miniaturisation is also described, since it allows high throughput of samples.

Retention behaviour of polar compounds using porous graphitic carbon with water-rich mobile phases

The retention factors of polar compounds (mono-, di- and trisubstituted aromatic derivatives) were measured on porous graphitic carbon (PGC), alkyl-modified silicas and an apolar copolymer (PRP-1) with water-methanol mobile phases. It was first shown that the mobile phase effects were similar with the three sorbents and that the comparison of retention factors extrapolated to aqueous mobile phases (k′w) could give information on stationary phase-solute interactions. The functional group contribution was examined. For aromatic derivatives containing hydrophobic substituents (alkyl and chloro groups), correlations with octanol-water partition coefficients were obtained for the three sorbents. For aromatic derivatives containing polar substituents, these correlations were obtained only for alkylsilicas and PRP-1. On PGC, the retention factor increased with increase in the number of polar substituents and was shown to depend on both the field and the mutual resonance effects of the different substituents on the aromatic ring. The results indicate that electronic interactions are more important than hydrophobic interactions in the retention mechanism of polar compounds. The parametrization of the polarity of the solutes, taking into account field and resonance effects, was carried out using local dipolar moments and the overall electron-excess charge density. The analyte retention factors could be predicted through correlation between log k′w and the electron-excess charge density.

Determination of some physicochemical parameters of microcystins (cyanobacterial toxins) and trace level analysis in environmental samples using liquid chromatography.

Some physicochemical parameters of three microcystin standards, known to be potent hepatotoxins produced by cyanobacteria, were determined using well defined chromatographic measurements. The logarithm of their retention factor on octadecylsilica (ODS) with water as the eluent, which is an estimation of the hydrophobicity of a molecule, was assessed at pH 7 at 3.9, 4.2 and 4.4 for microcystins-YR, -LR and -RR, respectively. Though being rather hydrophobic, microcystins also possess polar functions, namely carboxylic acids, amino and amido groups. The ionization of carboxylic groups occurs at pH values of 3.3-3.4. In environmental waters, microcystins are neutral or anionic. They are readily soluble in water, the solubility of microcystin-LR being higher than 1 g/l. Owing to their hydrophobicity and their polar functions, microcystins remain in the aqueous phase rather than being adsorbed on sediments or on suspended particulate matter. In a river water spiked with microcystins at 5 micrograms/l, only 10% was adsorbed on particles and 7% on the sandy sediment after three days. A method using solid-phase extraction on ODS followed by high-performance--or micro--liquid chromatography was optimized to detect microcystins at trace level in water. A clean-up was introduced to eliminate part of the interfering compounds coextracted during the sample percolation. Good recoveries (75-80%) were obtained. The method was linear, reproducible (with relative standard deviations ranging from 5 to 8%) and enabled the determination of microcystins at levels as low as 30 ng/l in drinking water and 100-200 ng/l in surface waters. The production of toxins by two strains of cyanobacteria was evaluated. Variations in the microcystin-LR content in the cells and in the medium of Microcystis aeruginosa PCC7806 were recorded over a five-week period. Toxin production was not correlated to the biomass but depended on the growth stage and was maximal at the end of the exponential growth phase. The release of toxin in water occurred essentially in old cultures where microcystin-LR was determined at concentrations of 170 and 280 micrograms/l in the media of M. aeruginosa PCC7806 and M. aeruginosa PCC7813, respectively. Other microcystins are likely to be synthesized by these strains. But owing to the lack of standards, mass spectrometric detection is required for further identification. This study points out the need of having other standards for water quality monitoring.