Laboratory calibration of a POCIS-like sampler based on molecularly imprinted polymers for glyphosate and AMPA sampling in water.

For more than 15 years, integrative passive sampling has been successfully used for monitoring contaminants in water, but no passive sampling device exists for strongly polar organic compounds, such as glyphosate. We thus propose a polar organic chemical integrative sampler (POCIS)-like tool dedicated to glyphosate and its main degradation product aminomethylphosphonic acid (AMPA), and describe the laboratory calibration of such a tool for calculating the sampling rates of glyphosate and AMPA. This passive sampler consists of a POCIS with molecularly imprinted polymer as a receiving phase and a polyethersulfone diffusion membrane. The calibration experiment for the POCIS was conducted for 35 days in a continuous water-flow-through exposure system. The calibration results show that the sampling rates are 111 and 122 mL day-1 for glyphosate and AMPA respectively, highlighting the potential interest in and the applicability of this method for environmental monitoring. The influence of membrane porosity on the glyphosate sampling rate was also tested. Graphical Abstract ᅟ.

Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser-induced fluorescence detection.

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and ß-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.

Synthesis and study of a molecularly imprinted polymer for specific solid-phase extraction of vinflunine and its metabolite from biological fluids.

A molecularly imprinted polymer (MIP) was synthesized in order to specifically extract vinflunine, an anticancer agent, and its metabolite (4-O-deacetylvinflunine) from bovine plasma and artificial urine by solid-phase extraction (SPE). Vinorelbine, a non-fluorinated analogue of vinflunine, was selected as a template for MIP synthesis. The selectivity of MIP versus the template (vinorelbine) and other alkaloids (catharanthine, vinblastine, vincristine, vinflunine and 4-O-deacetylvinflunine) was shown by a SPE protocol carried out with non-aqueous samples. A second protocol was developed for aqueous samples with two consecutive washing steps (AcOH-NH2 OH buffer (pH 7, I=10 mM)-MeOH mixture 95:5 v/v and ACN-AcOH mixture 99:1 v/v) and an elution step (MeOH-AcOH mixture 90:10 v/v). Thus, MIP-SPE of bovine plasma brought high recoveries, 81 and 89% for vinflunine and its metabolite, respectively. This protocol was slightly modified for artificial urine samples in order to obtain a good MIP/NIP selectivity; furthermore, elution recoveries were 73 and 81% for vinflunine and its metabolite, respectively. Repeatability was assessed in both biological matrices and RSD (%) were inferior to 4%. The MIP also showed a suitable linearity (r(2) superior to 0.99), between 0.25 and 10 µg/mL for plasma, and between 1 and 5 µg/mL for artificial urine.

Synthesis of a molecularly imprinted polymer for the solid-phase extraction of betulin and betulinic acid from plane bark.

INTRODUCTION: Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid-phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor-made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. METHODOLOGY: Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA- and AA-MIPs were compared with their non-imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP-SPE protocol. RESULTS: After SPE optimisation, the MAA-imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA-MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. CONCLUSION: This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology.

Capillary electrophoresis with dual detection UV/C4D for monitoring myrosinase-mediated hydrolysis of thiol glucosinolate designed for gold nanoparticle conjugation.

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate. Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few µL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.

Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen and its main metabolite from urine followed by high-performance liquid chromatography with UV detection.

A molecular imprinted polymer (MIP) has been synthesized in order to specifically extract tamoxifen, a nonsteroidal antiestrogen, and its metabolites from urine by solid-phase extraction (SPE) before HPLC-UV analysis. Clomiphene, a chlorinated tamoxifen analogue, was selected as template for MIP synthesis. Polymerisation was achieved by thermal polymerisation of methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile as porogen. The efficient elimination of the urinary matrix has been obtained by MIP-SPE but the elution recovery of tamoxifen was initially too low ( approximately 14%). This problem has been overcome following two ways. At first, a preliminary HLB-SPE of the urine has enabled to discard endogenous salts and to percolate an organic sample through the MIP cartridge. Extraction recoveries are equal to 56 and 74% for tamoxifen and 4-hydroxytamoxifen, respectively. Then, a second MIP has been prepared with styrene and MAA as functional co-monomers. Strong pi-pi interactions occurring between phenyl groups of styrene and tamoxifen promote rebinding of the analyte by the specific sites. The enhanced hydrophobic character of the imprinted polymer has enabled the direct percolation of urine through MIP-SPE and the easy elimination of endogenous salts from urine with only one aqueous washing step. HPLC-UV analysis has confirmed high extraction recoveries (85%) for tamoxifen and its metabolite with an enrichment factor of 8. This analytical protocol can selectively detect the presence of tamoxifen metabolites in urines and be useful as a proof of doping in competitive sports.

Preliminary recovery study of a commercial molecularly imprinted polymer for the extraction of glyphosate and AMPA in different environmental waters using MS.

A commercial molecularly imprinted polymer (MIP) dedicated to glyphosate (GLY) and its main metabolite, aminomethylphosphonic acid (AMPA), was lately assessed as "POCIS-like" sampler on mineral water. The obtained results were encouraging with 111 and 122 mL day-1 as sampling rates for GLY and AMPA, respectively. Therefore, before applying this passive sampler to environmental waters, the commercial phase was tested on different water matrices as a solid-phase extraction (SPE) device. The SPE protocol was carried on 250 mg of MIP with the following three steps: conditioning by Milli-Q water, loading of the sample (15 mL), and elution of the analytes by 4 mL 0.1 M HCl that were evaporated to dryness and recovered in 15 mL of the suitable solvent for analysis. This protocol was first applied to mineral water spiked by GLY and AMPA at environmental concentration levels (25-750 ng L-1). Analyses were carried out by ultra-performance liquid chromatography hyphenated to tandem mass after derivatization of GLY and AMPA by 9-fluorenylmethylchloroformate. The linear correlation between concentrations measured with and without SPE on MIP was proved.Furthermore, other extractions showed that high concentrations of metal ion interferents (lead(II), cadmium(II), and zinc(II)) in groundwaters did not reduce SPE performance of the MIP.Then, concentration assays were undertaken and brought noteworthy results, such as the recovery of 80% GLY and AMPA from groundwater spiked at 10 ng L-1 and concentrated 100 times. For this purpose, ion exclusion chromatography hyphenated to mass was applied without previous derivatization of the analytes. The same concentration factor and analytical method were applied to 100 ng L-1 spiked sea water with recoveries of 96% for GLY and 121% for AMPA.

Human neutrophil elastase inhibition studied by capillary electrophoresis with laser induced fluorescence detection and microscale thermophoresis.

Capillary electrophoresis-laser induced fluorescence (CZE-LIF) and microscale thermophoresis (MST) were used for the first time to study the inhibition of human neutrophil elastase (HNE). We recently studied HNE kinetics (Km and Vmax) by developing an in-capillary CZE-LIF assay based on transverse diffusion of laminar flow profiles (TDLFP) for reactant mixing. In this work, the former assay was adapted to monitor HNE inhibition. Two natural well known HNE inhibitors from the triterpene family, ursolic acid and oleanolic acid, were tested to validate the developed assay. Since the solubility of pentacyclic triterpenes in aqueous media where the enzymatic reaction will take place is limited, the effect of DMSO and ethanol on HNE was studied using microscale thermophoresis (MST). An agglomeration of the enzyme was revealed when preparing the inhibitor in 5% (v/v) DMSO. This phenomenon did not occur in the presence of ethanol. Therefore, ethanol was used as inhibitor solvent, at a limited percentage of 20% (v/v). In these conditions and after optimization of the TDLFP approach, the repeatability (RSD on migration times and peak-areas inferior to 2.2%) of the CZE-LIF assay and the sensitivity (LOQ of few nM) were found to be satisfactory for conducting inhibition assays. IC50 values for ursolic and oleanolic acid were successfully determined. They were respectively equal to 5.62±0.10µM (r(2)=0.9807; n=3) and to 8.21±0.23µM (r(2)=0.9887; n=3). Excellent agreement was found between the results obtained by CE and those reported in literature which validates the developed method. Particularly, the CE-based assay is able to rank HNE inhibitors relative to each other. Furthermore, MST technique was used for evaluating HNE interaction with the ursolic acid. Up to 16 capillaries were automatically processed to obtain in one titration experiment the dissociation constant for the HNE-ursolic acid complex. Ki was found to be 2.72±0.66µM (n=3) which is in excellent agreement with the value determined by CE enzyme inhibition studies (Ki=2.81µM) confirming the reliability of the developed CE assay and the competitive inhibition mode of ursolic acid.

Coated capillaries with highly charged polyelectrolytes and carbon nanotubes co-aggregated with sodium dodecyl sulphate for the analysis of sulfonylureas by capillary electrophoresis.

Sulfonylureas (SUs) are one of the most widely used herbicides to control weeds in crops. Herein, capillary electrophoresis (CE) was used to determine four sulfonylureas in natural waters, namely chlorsulfuron (CS), iodosulfuron methyl (IM), metsulfuron methyl (MSM) and mesosulfuron methyl (MSS). First of all, a bare silica capillary was chosen with 10mM of 1-butyl-3-methylimidazolium tetrafluoroborate (bminBF4) as electrophoretic buffer (pH 9.6) containing 2 mg L(-1) of surfactant-coated single-wall carbon nanotubes (SC-SWCNTs). A dramatic deviation in migration times was observed. Therefore, a poly(diallyldimethylammonium) chloride (PDADMAC) statically coated cationic capillary was used to improve repeatability and to alter the selectivity of the separation. The electroosmotic flow (EOF) measurement revealed that the SC-SWCNTs were strongly adsorbed at the surface of the PDADMAC coating even in the absence of the surfactant-coated nanotubes in the electrolyte buffer. Consequently, a stable strong cathodic EOF and excellent repeatabilities were obtained with relative standard deviations (RSDs) on migration times and on corrected peak areas below 0.9 and 1.5%, respectively. The separation of the SUs was conducted in only 6 min. No regeneration of the coating between analyses was necessary, and high peak efficiencies up to 173,000 theoretical plates were obtained. The bi-layer coating was subsequently used to analyze sulfonylureas in tap water, in several mineral waters as well as in underground waters spiked with SUs and directly injected into the CE capillary.

Capillary electrophoresis as a novel technique for screening natural flavonoids as kinase inhibitors.

Capillary electrophoresis (CE) was used for the first time to evaluate the inhibition activity of aglycone flavonoids (such as quercetin and isorhamnetin) and some of their glycosylated derivatives toward human kinases. The cyclin-dependant kinase 5 (CDK5/p25) and the glycogen synthase kinase 3ß (GSK3ß) were chosen since they are very promising biological targets for developing treatments against neurodegenerative diseases and cancer. In a previous work, we developed an in-capillary kinase CE assay where the capillary was used as an enzymatic nanoreactor in which the kinase, its substrate, adenosine 5'-triphosphate (ATP) and its potential inhibitor were mixed by using transverse diffusion of laminar flow profiles (TDLFP). The product adenosine 5'-diphosphate (ADP) was then detected at 254nm and quantified. In this work, this assay was improved to reduce, for the first time, the dilution effect commonly observed with the TDLFP approach. Under the new conditions established herein, IC50 values for quercetin, kaempferol and flavopiridol were successfully obtained and were in the same order of magnitude of those reported in the literature using the conventional assay using radioactive (33)P-ATP. It was shown that aglycone flavonoids have an inhibition activity more important than their glycosylated derivatives. CE was also proved to be very efficient for evaluating inhibition activity of complex samples such as crude extracts of sea buckthorn (SBT) berries obtained by solvent-free microwave extraction (SFME). This novel approach to combine SFME technique to a CE-based enzymatic assay is very interesting for evaluating the biological activity of natural material in a fast, simple, economic (no use of neither fluorescent nor radiometric labels) and green (no organic solvents) manner.

Convenient solid phase extraction of cephalosporins in milk using a molecularly imprinted polymer.

In this paper, a molecularly imprinted polymer (MIP) for cephalosporin molecules (cephalexin (CFL) and cephapirin (CFP)), was prepared by non covalent molecular imprinting approach and applied to solid phase extraction (SPE). For MIP synthesis, a tributylammonium cefadroxil salt (TBA-CFD) was used as template with methacrylic acid and ethylene glycol dimethacrylate as monomer and cross-linker, respectively, in acetone-methanol 92/8 (v/v) mixture. The selectivity of MIP versus non imprinted polymer (NIP) was confirmed for CFL, CFD and CFP in standard solutions as well as in milk samples. The efficiency of the synthesized MIP was evaluated by means of the application of the proposed MIP-SPE procedure to spiked milk samples previous to the HPLC method for the detection of cephalosporins. The MIP-SPE recoveries were higher than 60% for the three target analytes in spiked milk.

Analysis of urinary neurotransmitters by capillary electrophoresis: sensitivity enhancement using field-amplified sample injection and molecular imprinted polymer solid phase extraction.

Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H(3)PO(4)-LiOH (pH 4, ionic strength of 80 mmol L(-1)) as running buffer, 100 µmol L(-1) of H(3)PO(4) in methanol-water 90/10 (v/v) as sample solvent and 100 µmol L(-1) of H(3)PO(4) in water for the pre-injection plug. In these conditions, the linearity was verified in the 50-300 nmol L(-1) concentration range for DA, 3-MT and 5-HT with a determination coefficient (r(2)) higher than 0.99. The limits of quantification (10 nmol L(-1) for DA and 3-MT, 5.9 nmol L(-1) for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of neurotransmitters in urine, the presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE-UV method.Therefore, a solid phase extraction (SPE) on a dedicated imprinted polymer (MIP) was developed to extract specific neurotransmitters, catecholamines, metanephrines and indolamines, from urine. Matrix salts were thus discarded after sample extraction on AFFINIMIP™ Catecholamine & Metanephrine (100mg) cartridge. Therefore, lower limits of quantification were determined in artificial urine (46 nmol L(-1) for DA, 11 nmol L(-1) for 3-MT and 6 nmol L(-1) for 5-HT).The application of this protocol MIP-SPE/FASI-CE-UV analysis of neurotransmitters in human urine gave rise to electropherograms with a very good base line and signal to noise ratios above 15.

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: application to labetalol hydrochloride.

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C(4)D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol(+)) and its anionic counter-ion (Cl(-)) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software. This novel CE-C(4)D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time<10 min), precise (repeatability of migration times<0.7% and of corrected-peak areas < 3.3%; n=6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.

Selective solid-phase extraction of a triterpene acid from a plant extract by molecularly imprinted polymer.

A molecularly imprinted polymer (MIP) has been prepared by a thermal polymerisation method using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linking agent, chloroform as porogenic solvent and an oleanane triterpene compound (18-beta-glycyrrhetinic acid) as imprinted molecule (template). Equilibrium ligand binding experiments were done to assess the performance of the MIP relative to non-imprinted polymer (NIP). After optimisation of SPE protocol (CHCl3 as washing solvent and MeOH as elution solvent), successful imprinting was confirmed by comparison of the recoveries between NIP (5%) and MIP (97%) cartridges. The binding capacity of the MIP for 18-beta-glycyrrhetinic acid was determined to be 0.94 mg g(-1). Four structurally related oleanane triterpenes (18-alpha-glycyrrhetinic acid, oleanolic acid, echinocystic acid, erythrodiol) were selected to assess the MIP selectivity. Experimental data illustrated the influence of functional groups on the triterpene skeleton. The MIP was applied to the solid-phase extraction of triterpenoids from a plant extract prior HPLC analysis. However, CHCl3 was replaced by ACN during the washing step in order to suppress non-specific interactions due to polar matrix components. A selective extraction of 18-beta-glycyrrhetinic acid from hydrolyzed extract of liquorice roots was achieved with a good extraction yield (98%).