Selective and automated sample pretreatment by molecularly imprinted polymer for the analysis of the basic drug alfuzosin from plasma.

A molecularly imprinted polymer synthesized in dichloromethane, was evaluated for the selective extraction of a pharmaceutical compound from human plasma and integrated on-line with liquid chromatography. The target drug is an alpha-blocker called alfuzosin widely used for the treatment of benign prostatic hyperplasia. By a comprehensive approach of the retention mechanism, a selective extraction procedure was established by exploiting the development of electrostatic interactions between the target analyte and the selective support in LC compatible solvents. By applying this method to plasma, extraction recoveries close to 100% were obtained for alfuzosin while various pharmaceutical compounds currently found in biological fluids were not retained on the support. The high selectivity of the support coupled to the chromatographic system permitted an easy and fast analysis of the drug with a limit of quantification of 15 microg L(-1) by UV detection.

Towards comprehensive hydrocarbons analysis of middle distillates by LC-GCxGC.

The detailed characterization of middle distillates is essential for a better understanding of reactions involved in refining processes. Owing to a higher resolution power and an enhanced sensitivity, but especially to a group-type ordering in the chromatographic plane, comprehensive two-dimensional gas chromatography (GCxGC) offers unsurpassed characterization possibilities for petroleum samples. However, GCxGC fails to totally discriminate naphthenes from unsaturates occurring in hydrotreated diesel samples. This article aims at promoting the implementation of LC-GCxGC for the quantitative determination of hydrocarbon distribution in middle distillates, including naphthenes. In this configuration, liquid chromatography (LC) enables the separation of hydrocarbons into two fractions (viz., saturated and unsaturated) before the subsequent analysis of each fraction by GCxGC. In this paper, the choice of GCxGC conditions in order to achieve the separation and identification of hydrocarbons by chemical class is discussed; under these conditions, naphthenes are separated according to the number of saturated rings. For the first time, the presence of di-, tri-, and tetra-naphthenes resulting from the hydroconversion of aromatics can clearly be evidenced. A quantitative procedure for the determination of the distribution of hydrocarbons, including the distribution of naphthenes according to the number of saturated rings, is also proposed and discussed in detail. LC-GCxGC is found to provide an unequalled degree of information that will widely contribute to a better understanding of hydroconversion processes.

Robustness study of a reversed-phase liquid chromatographic method for the analysis of carboxylic acids in industrial reaction mixtures.

The robustness study of the reversed-phase liquid chromatographic method developed for the quantitative analysis of carboxylic acids is a real asset to prepare method transfer because it provides an indication of its reliability during routine use. Indeed, it was possible to predict the consequences of small variations in operating conditions on the responses. The design of experiments approach was applied to model the effects and interactions of a high number of factors varying simultaneously with a limited number of runs. First we identified the factors which potentially affect the chromatographic responses used for carboxylic acids quantitation: detection wavelength (lambda), column temperature (T), acetonitrile ratio in mobile phase (Me), duration of the plateau before the gradient (L) and gradient slope (S). Then we estimated the order of magnitude of realistic variations to assign factor levels. Finally a central composite design was carried out around the nominal conditions defined during method optimization. The statistical treatment of responses (retention factors, and concentrations) showed that the column temperature, the acetonitrile ratio in the mobile phase, the duration of the plateau before the gradient and the gradient slope were the most influent factors. The building of the robust domain from response-surfaces allowed us to give tolerance limits for the factors (216 nm< lambda <222 nm, 49.3 degrees C < T < 51.4 degrees C, 4.90% < Me < 5.18%, v/v, 4.5 min < L < 5.4 min, 9%< S <11%) for which the performances of the method were maintained.

Rapid polyelectrolyte-based membrane immunoassay for the herbicide butachlor.

Oppositely charged water-soluble polyelectrolytes were used in the developed membrane immunoenzyme assay for the herbicide butachlor. High-affinity and rapid binding between polyanion polymethacrylate and polycation poly(N-ethyl-4-vinylpyridinium) was applied to separate reacted and free immunoreactants. Competitive immunoassay format with peroxidase-labeled antigen was realized. The insoluble colored product of the peroxidase reaction was formed by bound labeled immune complexes and was reflectometrically detected. The assay combines short duration (15 min), high sensitivity (0.03 g/mL) and availability for out-of-laboratory testing. Different image processing algorithms were used to determine the herbicide content. Low variation coefficients of the measurements in the proposed quantitative assay, namely 4.8-9.0% for the range of antigen concentrations from 0.1 to 3.0 ng/mL, are evidence of the assay effectiveness. Possibility to control the butachlor content in mineral, artesian, and drinking water was demonstrated.

Retention mechanism of analytes in the solid-phase extraction process using molecularly imprinted polymers. Application to the extraction of triazines from complex matrices.

Two molecularly imprinted polymers (MIPs), prepared in dichloromethane with terbutylazine and ametryn as template molecules, were evaluated for the selective extraction of triazines from complex matrices. Various parameters affecting extraction recoveries on MIPs were studied in order to obtain an optimized extraction procedure allowing to reduce non-specific interactions. In order to test the selectivity of the MIPs, the same procedure was applied to the extraction of compounds possessing the same polarity and size as the triazines. By means of molecular modeling, the effects of the electric charge distribution and of the size of the molecules on the retention mechanism were studied. The value of capacity for terbutylazine MIP was also measured. At last, the high selectivity resulting from the use of MIPs was clearly demonstrated by their applications to the clean-up of grape juice and soil extracts spiked with triazines. In addition, the soil extract was cleaned-up by immunoextraction allowing the comparison of both approaches in terms of selectivity.

Immunoaffinity solid-phase extraction for pharmaceutical and biomedical trace-analysis-coupling with HPLC and CE-perspectives.

Immunoaffinity solid-phase extraction (SPE) technique is based upon a molecular recognition mechanism. The high affinity and the high selectivity of the antigen-antibody interactions allow the specific extraction and the concentration of the analytes of interest in one step. In pharmaceutical and biological fields, where most often matrices are complex and analytes at trace-levels, this approach constitutes a unique tool for fast and solvent-free sample preparation. This review presents a general description of this extraction technique and gives numerous examples of its applications in pharmaceutical and biomedical fields. It emphasizes the on-line coupling with chromatographic and electrophoretic separation techniques and introduces new developments. The future directions, especially with regards to the current development of analytical microsystems, are discussed.

Optimization of the class-selective extraction of triazines from aqueous samples using a molecularly imprinted polymer by a comprehensive approach of the retention mechanism.

Direct, selective solid-phase extraction of triazines from aqueous samples is presented using a molecularly imprinted polymer (MIP) made with terbutylazine as template molecule. After optimization of the steps of the procedure, 14 triazines including degradation products were studied and satisfactory extraction recoveries were obtained except for thiotriazines. By comparing results obtained with the terbutylazine MIP and a similar non-imprinted polymer, it was determined that retention was achieved via specific interactions except for hydroxyterbutylazine. Selectivity of the extraction procedure was also verified by applying the MIP for the extraction of phenylureas that were not retained on it. The effects of the charge distribution and of molecular volume of the triazines (obtained by molecular modeling) on the selectivity of interactions between the analytes and the MIP were studied. However, when the optimized procedure was applied to real samples, low extraction recoveries were obtained due to strong matrix effects: ion-exchange occurs between the carboxylate groups of the MIP and the ionic species of the sample, that prevents subsequent specific interactions. By introducing an acid wash step, the procedure was successfully applied for the class-selective extraction of triazines from industrial effluent and surface water samples. Finally, increased extraction recoveries were achieved for the polar degradation products of triazines by using a mixed-phase composed of a polymeric sorbent and the MIP.

Optimization, validation and comparison of various extraction techniques for the trace determination of polycyclic aromatic hydrocarbons in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection.

There is a need for a better characterization of sludges from wastewater treatment plants which are destined to be spread on agricultural lands. Inorganic pollutants are regularly controlled but organic pollutants have received few attention up to now. On this paper, we have been interested on the analysis of the 16 polycyclic aromatic hydrocarbons (PAHs) listed in the US Environmental Protection Agency (US EPA) priority list and more particularly of the six PAHs listed in the European community list (fluoranthene, benzo[b and k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene). The analysis step consists on liquid chromatography with both fluorescence and UV detections as described in the EPA Method 8310. As for the extraction step, several techniques such as supercritical fluid extraction, pressurized liquid extraction, focused microwave extraction in open vessels, Soxhlet and ultrasonic extractions are compared after optimization of the experimental conditions (solvent nature and quantity, temperature, pressure, duration, ... ) and validation with certified sludges. When optimized, these five extraction techniques are as much efficient with similar relative standard deviation. Whatever the extraction techniques used, the whole analysis protocol permits to quantify PAHs in the range of 0.09 to 0.9 mg/kg of dried sludges.

Toxicity of domoic acid in the marine mussel Mytilus edulis.

The neurotoxic, immunotoxic and genotoxic effects of domoic acid (DA) on the blue mussel Mytilus edulis were investigated by biomarkers, acethylcholinesterase (ChE) activity in gills, DNA fragmentation in digestive glands, vitality and phagocytosis activity of haemocytes in haemolymph of mussels. After intra muscular injection of DA at the concentrations ranging from 1-500 ng/g body weight (bw), no neurotoxic effect was detected within incubation times of 48 h and 7 d. The vitality of haemocytes remained in all mussels at the level of control samples within 48 h, and increased significantly after 7 d (P<0.05). At DA concentrations ranging from 1 to 100 ng/g bw haemocytes suggested a great phagocytosis activity, but no alteration in their number by both incubation times. By increasing DA concentration of 500 ng/g bw, the number of haemocytes doubled in 48 h without any change in phagocytosis activity. Primary DNA lesions in digestive glands of all injected mussels were determined in acute phase of poisoning within 48 h, and rapidly repaired after 7 d of incubation.

Picogram determination of "earthly-musty" odorous compounds in water using modified closed loop stripping analysis and large volume injection GC/MS.

"Earthy-musty" off-flavor problems in water samples are due to organic compounds present at the sub-part-per-trillion level. Numerous analytical methods such as purge and trap, liquid/liquid extraction, and closed-loop stripping analysis (CLSA) followed by GC/MS analysis have been used to determine these compounds. However, these methods offer poor sensitivity (detection limits of approximately 1 to 10 ng/L) when compared to the 20-30 pg/L of sensorial sensitivity. The purpose of this study was to develop a new method involving a modified CLSA preconcentration technique together with large volume injection GC/MS in order to attain analytical sensitivity equal to or better than olfactory sensitivity. For eight target compounds that cause taste and odor problems in water at trace levels, the method developed was linear in the 0.05-10 ng/L range and provided recoveries greater than 70% together with satisfactory repeatability. Detection limits as low as 15-30 pg/L were achieved, representing a 50-fold improvement in sensitivity as compared to current methods. The accuracy and sensitivity of the method were demonstrated in different aqueous matrixes, including raw surface water. The method was successfully applied to earthy-musty water samples that had remained unsolved by conventional techniques, thus proving its effectiveness.

Use of porous graphitic carbon coupled with mass detection for the analysis of polar phenolic compounds by liquid chromatography.

Phenolic compounds present in olive mill wastewater (OMW) need to be quantified because of their pollution capacity toward the environment. In the present study, six representative phenolic compounds of OMW were chosen to develop a LC-MS method. The high polarity of the compounds caused problems when using traditional reversed-phase liquid chromatography (RPLC). Consequently, a method was developed on another kind of chromatographic phase: Porous Graphitic Carbon (PGC) involving the use of a tetrahydrofuran (THF) gradient. The influence of THF as mobile phase in LC-MS coupling, which is not common practice, was evaluated. In Atmosperic Pressure Chemical Ionisation (APCI) in the negative ion mode, the presence of THF in the mobile phase did not degrade the MS signal of the target compounds in the conditions studied. On the contrary, an improvement was even observed when the percentage of THF increased. The proposed PGC-LC-MS method was selective and linear for the six phenolic compounds analysed with limits of quantification lower than 5 ppm in all cases. The precision was satisfactory (pooled RSD around 6%). The analyses of OMW matrix spiked sample confirmed the good performance of the proposed method.

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.

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.

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment.

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).

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.

Selective on-line immunoextraction coupled to liquid chromatography for the trace determination of benzidine, congeners and related azo dyes in surface water and industrial effluents.

A new extraction immunosorbent involving antigen-antibody interactions was coupled on-line to liquid chromatography for the selective extraction in aqueous samples of benzidine and congeners, widely used as intermediate compounds in the manufacturing of dyes and pigments. Due to the cross-reactivity of the antibodies for analytes with chemical structures closely related to that of the analyte used for immunization, the immunoextraction sorbent was shown to be able to extract aminoazobenzene and related azo dyes with good recoveries. The on-line coupling was optimized for the trace determination of benzidine, dichlorobenzidine, aminoazobenzene and some azo dyes with detection limits in the range 0.1 to 1 microgram/l. The high selectivity of the immunoextraction was shown by comparing the analysis of an industrial textile effluent obtained using precolumns packed either with a non-selective polymeric sorbent or with the anti-benzidine immunosorbent. In such complex samples, extraction and clean-up are achieved in the same step.

Isolation of priority polycyclic aromatic hydrocarbons from natural sediments and sludge reference materials by an anti-fluorene immunosorbent followed by liquid chromatography and diode array detection.

The selective isolation of PAHs from complex environmental mixtures was accomplished by means of a new methodology based on antigen--antibody interactions. This method consists on the extraction of PAHs from water samples onto an anti-fluorene immunosorbent (IS) followed by liquid chromatography with diode array detection. Environmental sediments and a sludge reference material containing PAHs were analyzed using this methodology in order to validate the performance of the IS for the cleanup procedure of such materials. Sediments were extracted by sonication with dichloromethane/methanol (2:1), and the extracts were brought to a volume of 100 mL of water in order to perform the extraction with the anti-fluorene IS. The reliability of the cleanup achieved by the IS was well demonstrated in the analysis of sediment and sludge complex samples containing the priority PAHs established by the U.S. EPA at concentrations varying from 56 micrograms/kg to 26 mg/kg. Results were compared to those obtained with conventional cleanup procedures showing a better selectivity for PAHs. The chromatograms presented a clear baseline allowing the determination and quantification of PAHs at the ppb level. Using immunosorbents, extraction, trace enrichment, and cleanup were accomplished in only one step.

Optimization and validation of solvent and supercritical-fluid extractions for the trace-determination of polycyclic aromatic hydrocarbons in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection.

There is a need for a better characterization of sludges from wastewater treatment plants which are destined to be spread on agricultural land. Inorganic pollutants are regularly controlled, but organic pollutants have received little attention up to now. The main problem for trace analysis of organic pollutants comes from the complexity of the various matrices of sludges, which depends on their origins. Therefore, methods described for soils and sediments cannot be directly applied to sludges which contain high amount of lipids. This paper provides a method for trace-determination of polycyclic aromatic hydrocarbons in the range of mg/kg of dried sludge including an extraction step, and an analysis step by liquid chromatography with fluorescence detection for sensitive quantification and UV-diode-array detection for confirmation. The extraction step was optimized by a selection of the solvent used for both Soxhlet and supercritical-fluid extraction and by setting the experimental conditions (temperature, pressure, modifier, etc.) used for SFE. Extraction recoveries were found similar and quantitative for the two procedures. However supercritical-fluid extraction appears to be a good alternative to Soxhlet extraction because of the consumption of less time and solvent. Detection limits were obtained in the range of 0.1-1 mg/kg of dried sludges, with possible confirmation by UV spectra. The whole method (extraction/quantification/confirmation) was validated using sludges which have been certified by the Community Bureau of Reference from Brussels (CRM No. 088). Application to the determination of PAHs in urban sludges is presented.

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.

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.