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Hitherto unknown chromophoric nucleosides are reported. This novel set of visibly coloured dye-labeled 5'-nucleosides, including 1,2,4,5-tetrazine, dicyanomethylene-4H-pyran, benzophenoxazinone, 9,10-anthraquinone and azobenzene chromophores, were prepared mainly under Cu-catalyzed azide-alkyne cycloaddition (CuAAC). The design criteria are outlined. Several derivatives possess in supplement a fluorescence property. The absorption and fluorescence spectra of all coloured nucleosides were recorded to study their potential as visible-range probes. Such nucleodyes are of great interest for future competitive lateral flow test MIP-based strips.

This study describes the synthesis and evaluation of different imprinted hydrogels using ribavirin as template molecule. Ribavirin serves as a model molecule because it possesses a broad-spectrum antiviral effect against RNA viruses, which are expected as emerging viruses. The choice of monomers enables to stabilize the pre-polymerization complex and to synthesize biocompatible polymers. Predictive studies as well as experimental works conclude similar results on best ribavirin:monomers ratios. Thus, materials exhibit high selective cavities toward ribavirin. These affinities allow to show release profiles drastically different from the non-imprinted ones at two temperatures. The imprinted materials show a sustained profile able to release antiviral for more than 24 h. The hydrogels obtained are biocompatible with model cells retained, human lung epithelial BEAS-2B cells. Cell viability is excellent and pro-inflammatory response is insignificant when imprinted polymers are incubated with cells. Finally, viral tests carried out on Influenza A infected lung cells show that imprinted delivery systems delivering 1 to 3 µg of antiviral have the same efficiency as a medium containing 30 µg mL-1 of active agent. As a very interesting result, the molecularly imprinted polymers as drug delivery systems allow to increase the local concentration of antiviral, to improve their delivery when its bioavailability is low.

International audience; Herein, we propose the synthesis of a microspherical imprinted hydrogel meant for the controlled release of a nucleotide, adenosine 5'-monophosphate (5'-AMP). Indeed, molecularly imprinted polymers-based (MIPs) materials possess remarkable selective molecular recognition ability that mimicks biological systems. MIPs have been used in numerous applications and hold great promise for the vectorization and/or controlled release of therapeutics and cosmetics. But, the conception of imprinted hydrogels-based drug delivery systems that are able to release polar bioactive compounds is explored weakly. Herein, the synthesis of imprinted hydrogel microbeads by inverse Pickering emulsion is detailed. Microspheres showed a large 5'-AMP loading capacity, around 300 mg·g-1, and a high binding capacity comparatively to the non-imprinted counterpart. The MIP had a thermo-responsive release behavior providing sustained release of adenosine 5'-monophosphate in an aqueous buffer simulating both human skin pH and temperature.

Modified nucleosides have become cornerstones of treatment for patients with cancer or viral infections. Moreover, they are excreted in the urine of cancer patients in high levels and are considered to be potential tumor biomarkers. ATP and its analogues play an important role in several functions of the cardiovascular system. Moreover, circulating nucleotides have therapeutic importance on the progression of metabolic disorders. Thus, their detection and quantization are important. To gain in selectivity, the design of artificial polymer receptors, so-called molecularly imprinted polymers (MIPs) that can selectively recognize nucleotide bases, has gained importance from the theoretical application point of view. We will initially review the articles that are interested in the development of MIPs for the recognition of nucleoside analogues. Then, the second part of the chapter will describe the work published on the use of MIPs in the field of separation techniques and sample pretreatment. The final part will summarize research where the MIP technology can improve the analytical selectivity of the sensors of all types.

SynopsisObjective The objective of this study was to synthesize a novel glucosamine-imprinted sorbent based on ionic and non-covalent dual approach to purify glucosamine from chicory root extracts. Methods The synthesis of the molecularly imprinted polymer was optimized in terms of choice of monomers, porogen, cross-linker and initiator to have the best recognition as possible for targeted molecule. The sorbent obtained was characterized by nitrogen sorption (BET), scanning electron microscopy (SEM) and solid-phase extraction (SPE) to plot adsorption isotherms. The selectivity of polymer between glucosamine and interfering salt as ammonium sulphate was calculated. Extraction procedure was optimized in terms of loading, washing and elution solvents, to have the best recovery of glucosamine. Compounds were analysed by HPLC-UV after chemical derivatization. Results The results showed that the optimal conditions of extracting glucosamine on this new type of sorbent were as follows: percolation of plant extract in EtOH/aqueous HCl pH 3, washing of cartridge with water and elution of compound of interest with aqueous acetic acid solution at 5%. The recoveries of glucosamine were around 53% and 70%, from aqueous standard solution and aqueous chicory roots extracts, respectively, on the molecularly imprinted polymer. And, only 11% and 7% of the ammonium sulphate were recovered from standard solution and chicory roots extract, respectively. Conclusion The use of the MIP as solid-phase extraction sorbent was able to extract preferentially glucosamine from structural analogues and ammonium salt. Assays on chicory roots extracts were carried out, and the MIP showed good results allowing the transfer methodology at semi-industrial scale for cosmetic companies. The optimized protocol of extraction of glucosamine allowed using only eco-friendly solvents, as ethanol, water and acetic acid.

A series of imprinted polymers targeting nucleoside metabolites, prepared using a template analogue approach, are presented. These were prepared following selection of the optimum functional monomer by solution association studies using 1 H NMR titrations whereby methacrylic acid was shown to be the strongest receptor with and affinity constant of 621 ± 51 L mol −1 vs. 110 ± 16 L mol −1 for acrylamide. The best performing polymers were prepared using methanol as porogenic co-solvent and although average binding site affinities were marginally reduced, 2.3 × 10 4 L mol −1 vs. 2.7 × 10 4 L mol −1 measured for a polymer prepared in acetonitrile, these polymers contained the highest number of binding sites, 5.27 μmol g −1 vs. 1.64 μmol g −1 , while they also exhibited enhanced selectivity for methylated guanosine derivatives. When applied as sorbents in the extraction of nucleoside derivative cancer biomarkers from synthetic urine samples, significant sample clean-up and recoveries of up to 90% for 7-methylguanosine were achieved.

Pseudouridine (?) is an important urinary cancer biomarker, especially in human colorectal cancer (CRC). Disclosed herein is the first ? molecularly imprinted polymer (?-MIP) material obtained from tailor-engineered functional monomers. The resulting MIP imprint exhibits a remarkable imprinting factor greater than 70. It is successfully used for the selective recognition of ? in spiked human urine. This selective functionalized material opens the route to the development of inexpensive disposable chemosensors for noninvasive CRC diagnosis and prognosis.

Three lipid fractions, namely the freely extractable fraction and those associated with humin and humic acid fractions, were obtained from the loamy soil of a carefully maintained long-term experiment located on Deffend ORE field, Poitiers, France. The analyses showed differences in molecular distribution, suggesting different sources and diagenetic states of the source material. Despite a major input of plant material to the soil organic matter, intensive bacterial activity was suspected. Most distributions suggested an increase in the microbial/terrestrial lipid ratio from the free to humin to humic fractions. Molecular evidence of fungal activity, especially in the top layer, was also found in the distributions of n-alkanes and n-alkanoic acids. In the surface horizon A1 alkanes were the major compounds, followed by n-alkanoic acids and sterols. The degraded horizons, poorer in organic matter, i.e. the A2 and B horizons, were dominated by long-chain (>C20) n-alkanoic acids with a strong even-over-odd predominance and C24 and C26 n-alkanols. Sterols had been removed from these horizons through degradation. A comparison of humic acid and humin composition on the basis of Py(methylation)-GC-MS showed that the two fractions produce partly similar pyrolysis products. Most prominent were molecules from plant and microbial carbohydrates, lignin building blocks and linear aliphatic (carboxylic acids, esters) and nitrogen compounds. The investigation showed that while low-molecular-weight soil lipids were highly dominated by compounds derived from the overlying vegetation, pyrolysis data from the corresponding high-molecular-weight fractions reflected the incorporation of microbial biomarkers into the humictype fractions.

An on-line liquid chromatography electrospray ionization mass spectrometry (MS) method was developed for the characterization of polymers of kappa- (extracted from Kappaphycus alvarezii), iota-, and hybrid iota-/nu-carrageenans (both extracted from Eucheuma denticulatum) enzymatically digested with specific carrageenase enzymes. Applying either CID MS/MS or in-source fragmentation mechanisms, the results demonstrated that none of the polymers of kappa- or iota-carrageenans existed with their ideal repeating units. On the polymer of kappa-carrageenan, the nonideal structures identified consisted of iota-neocarrabiose sulfate units. On the polymer of iota-carrageenan, the nonideal structures identified consisted of the following: (i) kappa-neocarrabiose sulfate units, (ii) iota-neocarrabiose sulfate units with an additional sulfate group, and (iii) iota-neocarrabiose sulfate units with an additional sulfate and a pyruvate acetal group. For both kappa- and iota-carrageenans, the nonideal structures were randomly distributed on the polymers. The method was then applied for the characterization of a hybrid polymer of iota-/nu-carrageenans, enzymatically digested with iota-carrageenase. The results did not reveal an ideal oligosaccharide of nu-carrageenan, suggesting that the iota-carrageenase enzyme could cleave only reduced "densities" of nu-carrageenan repeating units. In addition, information about the sequence of hybrid iota-/nu-carrageenans from E. denticulatum is deduced.

Enzymatically digested oligo-iota-carrageenans were separated with liquid chromatography, coupled to evaporative light scattering detection. As expected, compared to oligo-kappa-carrageenans, the additional sulphate group in the neocarrabiose unit of iota-carrageenans significantly modified the separation mechanisms on ion-exchange chromatography, porous graphitic carbon and ion-pair chromatography. The oligomers were then isolated and characterised off-line with electrospray ionisation mass spectrometry in positive-ion mode. The tetrasaccharide, hexasaccharide and octasaccharide that were identified were associated with protonated heptylamine molecules whose number depended on the number of sulphate groups.

Oligo-kappa-carrageenans participate as elicitors in the cell-cell recognition process in marine plants. Analytical methods can be usefully applied to gain insight into the biochemistry of these biological processes. Therefore, enzymatically digested oligomers of kappa-carrageenans have been separated and isolated on a Spherisorb ODS1 (250 x 4 mm i.d., particle size 5 microm) column using ion-pair liquid chromatography coupled with an evaporative light scattering detector. Heptylamine (5 mM, pH4) has been selected as the ion-pairing agent and MeOH as the organic modifier in a gradient mode. Overloading the column with 1mg of the mixture, the chromatographic mechanism presented adequate stability. The mobile phase of each isolated oligomer was evaporated and the residue was infused into an electrospray ionisation mass spectrometry (ESIMS) in positive-ion mode with 4:1 MeCN-water as mobile phase. Each ESIMS spectrum presented ions consisting of the oligomer attached with a number of heptylammonium ions depending on the molecule size. In addition, the different m/z values permitted direct detection of the oligomers in ESIMS positive-ion mode. The analytical method developed separated the oligomers up to dotriacontasaccharide.

International audience; This paper presents an overview of the main actions and projects of the ANR TECSAN "CancerSensor" during the 2011-2014 period. The project aims to develop and validate a quantitative, non-invasive therapeutic tool to detect selected urinary modified nucleosides as biomarkers of colorectal cancer chemotherapy and to monitor in fine the efficiency of the chemotherapy. A methodology has been developed to generate a bulk acoustic wave molecularly imprinted polymer sensor (SAW-MIP) for the detection of various nucleosides and nucleotides of interest. A rapid and sensitive mass spectrometry method has been also developed.

Sufentanil (SF) is a highly potent synthetic opioid (1) used extensively in anesthesia (2)(3). Most of the techniques proposed for SF analysis in plasma samples are based on the RIA method described by Michiels et al. (4). Some methods using gas chromatography–mass spectrometry (GC-MS) have been reported (5)(6)(7). Plasma concentrations of SF in the analgesic range are extremely low, and pharmacokinetic studies require the development of sensitive GC-MS assay methods. Reliable and reproducible isolation of SF from plasma is the most important and most critical step in pharmacokinetic analysis. Among different extraction methods currently used in pharmaceutics, the use of solid-phase extraction (SPE) has grown dramatically. However, according to the literature, liquid-liquid extraction (LLE) is the technique used most frequently for isolating morphinomimetic compounds (4)(5)(6)(7)(8), whereas only one method using SPE with RIA has been reported (9). We performed comparative studies on the isolation of SF and alfentanil (AF; used as an internal standard) from plasma using LLE and SPE methods, and extracts were assessed using GC-MS. The method was applied to drug monitoring in intensive care patients. SF citrate and AF hydrochloride were purchased from Janssen Pharmaceutica. Lyophilized plasma (Lyphochek®) was purchased from Bio-Rad Laboratories. Deionized water was prepared using a MilliQ® Water System (Millipore). Methanol (Uvasol® grade), dichloromethane (Uvasol grade), isoamyl alcohol (analytical grade), propan-2-ol (analytical grade), 13.2 mol/L ammonia, and sodium hydroxide (Normapur®) were purchased from Merck. n -Heptane (analytical grade), 18 mol/L sulfuric acid, and ammonium phosphate monobasic were purchased from Carlo Erba. Acetic acid and Sigmacote® were purchased from Sigma Aldrich. Oasis® MCX 60-mg SPE columns (Waters Corporation) were used, and the vacuum manifold for semiautomatic processing of the columns was purchased from Supelco. The Oasis MCX is a mixed-mode, …

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.

Enzymatically digested oligosaccharides of κ-carrageenans were separated on a porous graphitic carbon (PGC) column and characterised on-line by electrospray ionisation mass spectrometry (ESI–MS). Two different developing ions were applied. Among them ammonium hydrogencarbonate showed more eluting power as it should on normal anion-exchange stationary phases. The oligosaccharides were detected by ESI–MS as fully deprotonated oligosaccharides.

Carbohydrate-protein linkage region of proteoglycans is a key oligosaccharide structure because their sulphated and/or phosphorylated analogues control the biosynthesis of glucosaminoglycans or galactosaminoglycans. Therefore, synthesised sulphated and/or phosphorylated analogues were characterised by tandem mass spectrometry in the negative-ion mode. Results demonstrated that the product ion profile was characterised by glycosidic and cross-ring cleavages depending on the position and the type of the charged group (sulphate, phosphate or carboxylate). When the above compounds were sulphated and phosphorylated, the ion found at m/z 79 was the only one that demonstrated a phosphate group on the structure. The data also suggested that when a sodium cation was present in a sulphated and phosphorylated structure, the phosphate group in most cases was neutralised by the sodium cation, and therefore cleaved off the molecule, while the sulphate group was carrying the negative charge.

A new methylated β-cyclodextrin with a low degree of substitution was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) with evaporative light scattering detection. Using α-cyano-4-hydroxycinnamic acid as the matrix and the thin layer method as the deposition procedure, MALDI-TOF-MS revealed that the mixture was composed of CDs bearing from 2 to 8 methyl groups with an average degree of substitution (DS) of 0.7 (i.e. 0.7 methyl groups per glucopyranose unit). Using a Purospher Star RP-18 endcapped column with acetonitrile–water mobile phase in gradient elution mode, HPLC was employed at analytical scale to obtain a chromatographic fingerprint of the crude mixture and at semi-preparative scale to fractionate it. MALDI-TOF-MS of these fractions revealed that the overall retention of the different derivatives, which depicts their polarity, was mainly driven by the DS and increased with the number of methyl groups on the CD moiety.

International audience; Enzymatically digested oligosaccharides of kappa-, iota- and hybrid iota/nu-carrageenans were analysed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the negative-ion mode. nor-Harmane was used as matrix. Depending on the stock concentration and the laser intensity applied, the oligosaccharides exhibited losses of sulphate units (neutralised by the Na+ ion, and thus non-stable), leaving the primary backbone structure in most cases with only the deprotonated sulphate groups (carrying the negative charge, stable). This meant that kappa- and iota-oligosaccharides could not be easily distinguished from one another since they share the same primary backbone structure. However, for the hybrid iota/nu-oligosaccharides the primary backbone structure could be identified since the nu-carrageenan repeating unit differs from that of the kappa/iota-carrageenan unit. For all types of oligosaccharides, the results indicated cleavage of an anhydrogalactose unit from the non-reducing end. Specifically, for the hybrid oligosaccharides of iota/nu-carrageenans, this type of fragmentation means that the nu-carrageenan unit is not positioned on the non-reducing end of the hybrid oligosaccharides. Dehydration reactions, and exchange reactions of Na+ with K+ and Ca2+, were also observed.

The antioxidant activity of propofol, a widely used anesthetic, has previously been demonstrated, but no study has focused on propofol metabolites although propofol undergoes extensive metabolism. In the present study, the antioxidant properties of propofol and its metabolites were studied by measuring malondialdehyde (MDA) produced from lipid peroxidation by microsomes triggered with several free radical generating systems. True MDA determination was performed using a specific high performance liquid chromatography technique. Gas chromatography-isotope ratio mass spectrometry methodology was also used to assess the antioxidant action in a homogeneous aqueous environment. Propofol, 2,6-di-isopropyl-1,4-quinol (1,4-quinol) metabolite and 3,5-di- tert-butyl-4-hydroxytoluene markedly inhibit lipid peroxidation at concentrations lower than 5 microM. The binding of the glucuroconjugated moiety to either one of two hydroxyl groups of 1,4-quinol lowers the radical scavenging activity. Propofol glucuronide did not exert any radical scavenging activity except when peroxidation was induced with tert-butylhydroperoxide. Our data demonstrate that propofol and its metabolites inhibit lipid peroxidation at concentrations similar to those measured in human plasma during anesthesia. Their antioxidant efficiency is influenced by several factors, including the type of radical initiator involved and the site of radical production.

The stability of sufentanil in human plasma kept under various storage conditions was investigated. Extraction was performed using solid phase extraction with new mixed-mode cation exchange Oasis MCX columns; quantification was carried out using gas chromatography equipped with a mass spectrometry detector. When plasma was left at 4 degrees C in nonsilanized tubes, concentrations of sufentanil decreased significantly during the first hour. In plasma samples kept at -25 degrees C for 8 hours in nonsilanized glass tubes, a significant decrease of sufentanil concentrations was found, with an average loss of 10.1% of the initial concentration. A significant decrease occurred when plasma was kept in silanized glass tubes for 12 hours at -25 degrees C. The current study emphasizes the importance of sampling and storage conditions for an accurate determination of sufentanil concentration in plasma.