Molecularly imprinted polymer for the selective extraction of cocaine and its metabolites, benzoylecgonine and ecgonine methyl ester, from biological fluids before LC-MS analysis.

Considering the important complexity of biological samples, a molecularly imprinted polymer (MIP) was applied to the selective extraction of cocaine and its two main metabolites, benzoylecgonine and ecgonine methyl ester from biological samples. The MIP was imprinted with cocaine and it was synthesized in acetonitrile with methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The selectivity of the MIP was first assessed for the three target analytes in acetonitrile with recoveries higher than 80% on the MIP and lower than 30% on the non-imprinted polymer (NIP). The MIP was then evaluated for the selective extraction of these targets from real aqueous media, i.e. serum and urine samples. The pH adjustment of the sample as well as the optimization of the washing step led to a very selective extraction of cocaine from these media. A LOQ of 0.5ng/mL was obtained for cocaine in urine. Concerning cocaine metabolites, benzoylecgonine and ecgonine methyl ester, they were first extracted from urine by liquid-liquid extraction and the resulting extract was purified on the MIP. The results obtained with the MIP as compared to the LLE alone showed the great potential of the MIP extraction for the clean-up of the biological matrix. This procedure was tested for the extraction of the analytes from urine samples, leading to a very selective protocol with LOQs of 0.09ng/mL, 0.4ng/mL and 1.1ng/mL for cocaine, benzolecgonine and ecgonine methyl ester respectively in urine samples.

Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: an efficient tool in the control of natural steroid hormones abuse in cattle.

The use of anabolic substances to promote growth in livestock is prohibited within the European Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected as the most relevant matrix and, due to its relatively high content of potential interferents, its preparation before analysis is considered as a key step. In this context, the use of a selective sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17ß-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC-MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries (upper 65% for the 12 target steroids) with sufficient purification for gas chromatography-mass spectrometry (GC-MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS) requires a higher degree of purity of the target compounds, which can be reached by the SFC-MIP protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of (13)C/(12)C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy.

Synthesis, configuration assignment, and simultaneous quantification by liquid chromatography coupled to tandem mass spectrometry, of dihydroanatoxin-a and dihydrohomoanatoxin-a together with the parent toxins, in axenic cyanobacterial strains and in environmental samples.

We have synthesized cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a using a short synthetic route. The relative configuration of N-tert-butoxycarbonyl-cis-dihydroanatoxin-a was determined by X-ray crystallography, while that of N-tert-butoxycarbonyl-trans-dihydroanatoxin-a was confirmed by epimerization leading to the cis-diastereoisomer. The relative configuration of N-tert-butoxycarbonyl-trans- and cis-dihydrohomoanatoxin-a was inferred from their NMR spectra. Using an optimized LC-MS/MS analytical method and pure standards we have simultaneously determined anatoxin-a, homoanatoxin-a and their dihydroderivatives in axenic strains of cyanobacteria and in environmental samples from the Tarn River, France. However, in these analytical conditions, the cis- and trans-dihydroanatoxin-a and cis- and trans-dihydrohomoanatoxin-a could not be separated. In axenic strains, the dihydroderivatives represented less than 3% of the total toxin content, while in field samples dihydroanatoxin-a represented from 17% to 90% of the total toxin content. Thus, the reduction of anatoxin-a to dihydroanatoxin-a is predominant in the environment. The ratio of anatoxin-a concentration over that of homoanatoxin-a in axenic strains was variable, and among the eight strains studied we found three exclusive anatoxin-a producers and five producers of homoanatoxin-a and anatoxin-a, the latter representing from 0.5% to 2.0% of the total. In the strains studied, we have amplified by PCR, and sequenced the region of anaG coding for the methylation domain proposed to be responsible for the formation of homoanatoxin-a. The sequences showed at least 88% identity and we could not relate the toxin profile of the strains to the sequence of the methylation domain.

Three-phase hollow fiber liquid-phase microextraction of organophosphorous nerve agent degradation products from complex samples.

Degradation products of chemical warfare agents are considered as important environmental and biological markers of chemical attacks. Alkyl methylphosphonic acids (AMPAs), resulting from the fast hydrolysis of nerve agents, such as sarin and soman, and the methylphosphonic acid (MPA), final degradation product of AMPAs, were determined from complex matrices by using an emergent and miniaturized extraction technique, the hollow fiber liquid-phase microextraction (HF-LPME), before their analysis by liquid chromatography coupled to mass spectrometry (LC-MS). After studying different conditions of separation in the reversed phase LC-MS analysis, the sample treatment method was set up. The three-phase HF-LPME was carried out by using a porous polypropylene (PP) hollow fiber impregnated with 1-octanol that separates the donor and acceptor aqueous media. Various extraction parameters were evaluated such as the volume of the sample, the effect of the pH and the salt addition to the sample, the pH of the acceptor phase, the extraction temperature, the stirring speed of the sample, the immersion time in the organic solvent and the time of extraction. The optimum conditions were applied to the determination of MPA and five AMPAs in real samples, such as surface waters and urine. Compounds were extracted from a 3 mL acidified sample into only 6 µL of alkaline water without any other pretreatment of the complex matrices. Enrichment factors (EFs) higher than 170 were obtained for three less polar AMPAs. Limits of quantification (LOQs) in the 0.013-5.3 ng mL(-1) range were obtained after microextraction of AMPAs from river water and in the range of 0.056-4.8 ng mL(-1) from urine samples with RSD values between 1 and 9%.

Synthesis and characterization of molecularly imprinted polymers for the selective extraction of cocaine and its metabolite benzoylecgonine from hair extract before LC-MS analysis.

A molecularly imprinted polymer (MIP) was synthesized and evaluated for the selective extraction of cocaine (COC) and its main metabolite benzoylecgonine (BZE) in hair extracts. To this end, a screening of different conditions of synthesis was performed by changing the nature of the crosslinker, and the functional monomer and also by changing polymerization's initiation mode. The selectivity of the different MIPs was evaluated by comparing the retention of COC and BZE between the MIP supports and also compared to a non-imprinted polymer for each. All the supports were selective for one or both molecules, but, the best results in terms of selectivity and retention were obtained for a MIP using methacrylic acid as functional monomer, ethyleneglycol dimethacrylate as crosslinker, and a photochemical initiation. An optimized procedure in acetonitrile media was developed for the selective extraction of COC and BZE with a recovery close to 80% for both molecules from the MIP. The capacity of the MIP for COC retention was also evaluated, and MIP showed a specific capacity of 8.96µmolg(-1). Finally, the potential of this material for sample clean-up was demonstrated by the selective extraction of both COC and BZE from acetonitrile hair extracts spiked at the cutoff value for COC in hair analysis. By the selective purification with the MIP, a limit of quantification inferior to 0.07 ng mg(-1) of hair was reached for both molecules.

Novel extraction supports based on immobilised aptamers: evaluation for the selective extraction of cocaine.

A new kind of selective sorbent based on the use of aptamers and dedicated to the selective solid phase extraction was developed. Cocaine aptamer was chosen as model aptamer to demonstrate the feasibility of this material and to provide a complete evaluation of the synthesized sorbent. The effect of different parameters such as the nature of the immobilisation support (silica, agarose), the type of immobilisation (covalent or non-covalent) and the length of the spacer arm (C(6) or C(12)) were studied. Therefore, various oligosorbents based on different immobilisation strategies were synthesized and characterised by estimating the extraction recovery and the capacity of cocaine and the binding efficiency of aptamers. Control supports without immobilised aptamers were simultaneously studied in parallel to assess the selectivity brought by the oligosorbents. The oligosorbent based on CNBr-activated sepharose showed the best performances with an extraction recovery for cocaine of 90% while 6% was obtained on the control sorbent. The high selectivity brought by the oligosorbent was then illustrated by applying the oligoextraction followed by LC/MS analysis to a post-mortem blood (cocaine overdose). Results were compared to those resulting from a conventional protein precipitation procedure. The presence of co-extracted interfering compounds was strongly reduced with the treatment by oligoextraction. A limit of quantification of 0.5 ng/mL was obtained that is largely lower than the concentration found after a single intake of cocaine.

New extraction sorbent based on aptamers for the determination of ochratoxin A in red wine.

A new solid phase extraction method based on aptamers, an oligosorbent, was developed and applied to the determination of ochratoxin A (OTA) from red wine. Two solid supports were chosen to immobilize OTA aptamer by covalent binding (cyanogen bromide-activated sepharose) or noncovalent binding (streptavidin-activated agarose). The resulting oligosorbents were evaluated in terms of retention, selectivity, and capacity. To assess the selectivity of the resulting oligosorbents, control supports made only of a solid support without immobilized aptamers were simultaneously studied. After optimization of the selective extraction procedure, extraction recoveries close to 100% were obtained on both materials. No retention was observed on the control supports. A similar capacity was also found for both oligosorbents. However, the immobilization by covalent bonding appeared more robust for the determination of OTA in the wine. A conventional sorbent and an immunoaffinity column were also applied to the determination of OTA in red wine to compare the potential of the various approaches for the treatment of such complex samples.

Selective extraction of nitroaromatic explosives by using molecularly imprinted silica sorbents.

Two molecularly imprinted silicas (MISs) were synthesized and used as selective sorbents for the extraction of nitroaromatic explosives in post-blast samples. The synthesis of the MISs was carried out with phenyltrimethoxysilane as monomer, 2,4-dinitrotoluene (2,4-DNT) as template and triethoxysilane as cross-linker by a sol-gel approach in two molar ratios: 1/4/20 and 1/4/30 (template/monomer/cross-linker). Non-imprinted silica sorbents were also prepared following the same procedures without introducing the template. An optimized procedure dedicated to the selective treatment of aqueous samples was developed for both MISs for the simultaneous extraction of the template and other nitroaromatic compounds commonly used as explosives. The capacity of the MISs was measured by the extraction of increasing amounts of 2,4-DNT in pure water and is higher than 3.2 mg/g of sorbent for each MIS. For the first time, four nitroaromatic compounds were selectively extracted and determined simultaneously with extraction recoveries higher than 79%. The potential of these sorbents was then highlighted by their use for the clean-up of post-blast samples (motor oil, post-mortem blood, calcined fragments, etc.). The results were compared to those obtained using a conventional sorbent, thus demonstrating the interest of the use of these MISs as selective sorbents.

Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis.

Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 microg/mg and 0.29 microg/mg, respectively) and cylindrospermopsin (0.10 microg/mg and 0.11 microg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 +/- 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.

Development of imprinted materials for the selective extraction of nitroaromatic explosives.

Molecularly imprinted sorbents were synthesized and used as selective extraction sorbents for the analysis of nitroaromatic explosives. Their synthesis by radical polymerization using organic monomers and by sol-gel approach using organosilanes was considered to develop a selective sorbent. The sol-gel approach with phenyltrimethoxysilane (PTMS) as monomer and 2,4-dinitrotoluene (2,4-DNT) as template gave the most promising results. An optimized procedure adapted to the selective treatment of aqueous samples was then developed and applied to various target explosives. For the first time four nitroaromatic compounds were retained on the molecularly imprinted silica (MIS) with extraction recoveries between 29% and 81%, while only low recoveries were obtained on the non-imprinted sorbent, thus highlighting the high degree of selectivity. The MIS was then used for the clean-up of a sample containing motor oil spiked with 2,4-DNT and 2,4,6-trinitrotoluene (2,4,6-TNT). The results were compared with those obtained using a conventional sorbent (Oasis HLB). The cleanest chromatogram obtained using the MIS emphasized the high potential of the MIS as selective sorbent.

Solid-phase extraction using molecularly imprinted polymers for selective extraction of a mycotoxin in cereals.

The aim of this work was to develop a method for the clean-up of a mycotoxin, i.e. Ochratoxin A (OTA), from cereal extracts employing a new molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE) and to compare with an immunoaffinity column. A first series of experiments was carried out in pure solvents to estimate the potential of the imprinted sorbent in terms of selectivity studying the retention of OTA on the MIP and on a non-imprinted polymer using conventional crushed monolith. The selectivity of the MIP was also checked by its application to wheat extracts. Then, after this feasibility study, two different formats of MIP: crushed monolith and micro-beads were evaluated and compared. Therefore an optimization procedure was applied to the selective extraction from wheat using the MIP beads. The whole procedure was validated by applying it to wheat extract spiked by OTA at different concentration levels and then to a certified contaminated wheat sample. Recoveries close to 100% were obtained. The high selectivity brought by the MIP was compared to the selectivity by an immunoaffinity cartridge for the clean-up of the same wheat sample. The study of capacity of both showed a significant higher capacity of the MIP.

Selective sample pretreatment by molecularly imprinted polymer for the determination of LSD in biological fluids.

For the first time, a molecularly imprinted polymer (MIP) was synthesized by a noncovalent imprinting approach for the selective extraction of an illicit drug, LSD, from hair and urine samples. For the synthesis of MIP, an analog of LSD, was taken as a dummy template, methacrylic acid as a functional monomer, and ACN as a porogen solvent. The MIP was used for offline extraction before HPLC-MS analysis. By studying the interactions taking place between the LSD and the MIP, a selective procedure was established in organic media and applied to hair samples. By this way, 0.1 ng/mg of LSD was successfully detected in hair with 82% of extraction recovery. A low retention was also obtained on the control polymer (only 9%). This procedure was then modified to obtain a selective extraction in aqueous media for the determination of LSD in urine samples. The comparison with a conventional C18 clearly demonstrated the selectivity brought by the MIP to the determination of LSD in urine. LSD was easily detected in urine at only 0.5 ng/mL with 83% of extraction recovery on the MIP and 11% on the NIP. An LOQ of 0.2 pg/mL was estimated in urine samples.

Determination of cocaine in human plasma by selective solid-phase extraction using an aptamer-based sorbent.

A complete characterization is presented of a highly selective solid-phase extraction (SPE) sorbent which exploits the properties of aptamers. An oligosorbent based on aptamers immobilized on a solid support was synthesized and tested for the selective extraction of cocaine from human plasma. Anticocaine aptamers were immobilized to CNBr-activated Sepharose, and an extraction procedure was developed in pure media. Specific retention of cocaine on the oligosorbent was demonstrated, and the capacity of the support was determined. This oligosorbent was then applied to the selective extraction of cocaine from plasma at a concentration of 0.4 mg L(-1), i.e., corresponding to the plasma concentration reached after an intake of a single dose of cocaine. Extraction recovery close to 90% was obtained. Moreover, interfering compounds that perturbed cocaine quantification when using a standard SPE sorbent were not retained on the oligosorbent, thus allowing fast and reliable analyses of plasma samples with an estimated limit of detection of 0.1 microg mL(-1).

Role of molecularly imprinted polymers for selective determination of environmental pollutants--a review.

The molecularly imprinted polymers (MIPs) are synthetic polymers possessing specific cavities designed for a target molecule. By a mechanism of molecular recognition, the MIPs are used as selective tools for the development of various analytical techniques such as liquid chromatography, capillary electrochromatography, solid-phase extraction (SPE), binding assays and biosensors. This review describes the application of MIPs to the determination of environmental pollutants in these different analytical approaches with a special emphasis on their potential as selective SPE sorbent for the selective extraction of target analytes from complex matrices.