Performance evaluation of a MIP for the MISPE-LC determination of p-[18F]MPPF and a potential metabolite in human plasma.

Within the family of serotonin (5-HT) receptors, the 5-HT1A subtype is particularly interesting as it may be involved in various physiological processes or psychological disorders. The p-[18F]MPPF, a highly selective 5-HT1A antagonist, is used for in vivo studies in human or animal by means of positron emission tomography (PET) [1]. In order to selectively extract p-[18F]MPPF and its main metabolites from plasma, molecularly imprinted polymer (MIP) was prepared against these compounds by using the p-MPPF as template. For the control of the selectivity, non-imprinted polymer (NIP) was also synthesized without template. The MIP sorbent, packed in disposable extraction cartridges (DECs), was then evaluated as molecularly imprinted solid-phase extraction (MISPE) prior to the LC determination. The conditions of extraction were evaluated in order to obtain the highest selective retention of the p-[18F]MPPF and its metabolites on this MIP. The MIP selectivity was exploited in the loading and washing steps by adjusting the pH of plasma samples at a suitable value and by selecting mixtures for the washing step to limit the contribution of non-specific interactions. Other important parameters involved in the conditioning and elution steps were also studied. Finally, a pre-validation was carried out with optimal extraction conditions to demonstrate the performance of this MISPE-LC method as a generic method in the context of evaluation of new MISPE for p-[18F]MPPF and its potential for metabolites extraction from human plasma.

Molecularly imprinted polymers for the determination of organophosphorus pesticides in complex samples.

Organophosphorus compounds constitute an important class of pesticides whose the toxicity of which arises from the inhibition of the acetylcholinesterase enzyme. They exhibit a wide range of physico-chemical properties, thus rendering their determination in complex oil samples particularly difficult. To facilitate their analysis at the trace level in various samples (environmental waters, soils, vegetables…), molecularly imprinted polymers (MIPs) that are synthetic polymers possessing specific cavities designed for a target molecule have been prepared. Often called synthetic antibodies, MIPs can replace antibodies in different application fields. Indeed, as immunosorbents, MIPs can be used as selective sorbents for the solid phase extraction of target analytes from complex matrices or as recognition elements in sensors. Their synthesis, characterization and use as selective sorbent for the selective recognition of organophosphorus pesticides have been already largely described and are summarized in this review.

Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry.

A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).

Molecularly imprinted polymer for analysis of zidovudine and stavudine in human serum by liquid chromatography-mass spectrometry.

A molecularly imprinted polymer (MIP) using zidovudine (AZT) as template and methacrylic acid as monomer was prepared. The synthesis of the MIP was performed in acetonitrile. The synthesized material was then tested for the solid-phase extraction of AZT from different media (pure organic solvents and hydro-organic mixtures). An optimised procedure was developed for the selective extraction of AZT with a recovery of 96% using the MIP and only 3% on a non-imprinted polymer used as control polymer. A specific capacity of 0.2 micromol g(-1) was determined. The specificity of the MIP was evaluated by studying the retention behaviour of two others nucleoside analogues. The feasibility of the MIP to selectively extract AZT and stavudine (d4T) from human serum was also demonstrated with recoveries of 80 and 85% respectively. The lower limit of quantification (LLOQ) and the lower limits of detection (LLOD) for AZT were 5.10(-7) and 10(-7) M respectively.

Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass spectrometry detection for quantitative analysis of cytochrome c.

An in-line procedure for protein analysis using a trypsin-based immobilized enzymatic reactor (IMER) coupled to LC-MS/MS has been developed. Various IMERs were synthesized and characterized by estimating the digestion yield of a pattern peptide in UV detection. Laboratory-made IMERs were optimized by studying the effect of different parameters as the nature of the functionalized immobilization support (silica, agarose), the amount of immobilized trypsin and the binding density. The potential of the laboratory-made IMERs were compared with a batch digestion and with a commercial trypsin-based IMER. The laboratory-made IMER based on CNBr-activated Sepharose showed the best performances in terms of digestion yields, digestion time, price and repeatability (RSD<4%). Cytochrome c was then digested on this IMER and used in-line with LC-MS. The target protein was easily recognized by the Mascot database until 17pmol injected.

Molecularly imprinted polymer applied to the determination of the residual mass of atrazine and metabolites within an agricultural catchment (Brévilles, France).

Atrazine, desethyl-atrazine and desisopropyl-atrazine have been measured in the soils of Brévilles watershed. Pressurised liquid extraction (PLE) technique was used for extraction followed by purification with terbutylazine molecularly imprinted polymers. This clean-up procedure allowed to remove interfering compounds from the sample extracts. Thus making easier the analyses by reversed phase liquid chromatography coupled with ion trap tandem mass spectrometry. This selective sample treatment for soil extracts allowed limit of quantification (LOQ) of 0.03 ng/g for atrazine and 0.05 ng/g for metabolites. The concentrations in soil samples ranged from 7.1 ng/g to < LOQ for atrazine and from 2.5 ng/g to < LOQ for metabolites. The total cumulated mass in the top 60 cm of the soil of the watershed was estimated at 1.4, 0.52 and 0.25 kg for atrazine, desethyl-atrazine and desisopropyl-atrazine, respectively. A fraction of this mass available for leaching could generate water infiltrating with concentrations higher than the drinking water limit, 7 years after the last application of atrazine.

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.

An interlaboratory study to evaluate potential matrix reference materials for herbicides in water.

Matrix reference materials (MRM) are essential tools for the validation of analytical protocols. Nowadays, there are no such materials for the determination of herbicides in water. Pesticides stored in acetonitrile and stored on solid phase extraction (SPE) cartridges previously percolated with a water sample spiked with triazines and phenylureas have proven to be good candidates for reference materials because of their satisfactory stability under appropriate temperature conditions. To evaluate the behaviors of these materials containing pesticides and to be analyzed by liquid chromatography, a collaborative study including 15 laboratories has been organized. Observed reproducibility on candidate materials after the removal of extreme results was 16.1% for the vials with pesticides in acetonitrile (at around 0.125 mg/L) directly analyzed, 29.2% for a water sample spiked with the pesticides (at around 0.5 microg/L) analyzed after preconcentration on a cartridge and 26.7% for the cartridges previously percolated with a water containing the pesticides (250 mL at around 0.5 microg/L for each pesticide) analyzed after elution. Such dispersion values are quite compatible with the requirement of a further certification for such materials.

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.

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.

[Liver transplantation in a patient with hemophilia A and end stage liver failure]. / Transplantation hépatique chez un hémophile A atteint d'une insuffisance hépatocellulaire sévère.

We report the case of a 50 year-old man factor VIII deficient haemophiliac and hepatitis C cirrhosis. The patient underwent orthotopic liver transplantation because of episodes of variceal bleeding and encephalopathy. He received factor VIII replacement therapy perioperatively. Factor VIII returned to normal within 24 hours postoperatively and factor VIII replacement was stopped. Liver transplantation can be considered as definitive therapy for haemophilia.

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.

Solid-phase extraction for multiresidue analysis of organic contaminants in water.

To overcome the limitations of the detection systems associated with gas or liquid chromatography, a sample pretreatment is required with the objective to provide a sample fraction enriched with all the target analytes and as free as possible from other matrix components. There is now no doubt that solid-phase extraction (SPE) has now become the method of choice for carrying out simultaneously the extraction and concentration of many compounds in aqueous samples. Many recent applications of SPE to multiresidue analysis are reviewed with an emphasis on the importance of the choice of the sorbent and of the sample volume. SPE is particularly well adapted to multiresidue analysis including compounds from a wide range of polarity or characterized by various physico-chemical properties. However, SPE is not completely free from practical problems inherent to the nature of the compounds or to the coupling to the chromatographic systems. Many examples are reported to illustrate these problems which can in most cases be circumvented. New developments in SPE are also reviewed.

Okadaic acid production from the marine benthic dinoflagellate Prorocentrum arenarium Faust (Dinophyceae) isolated from Europa Island coral reef ecosystem (SW Indian Ocean).

Okadaic acid was isolated from a strain of Prorocentrum arenarium Faust (Prorocentrales, Dinophyceae) collected from Europa Island (40 degrees 22'E, 22 degrees 20'S, SW Indian Ocean). The presence of okadaic acid in the algal extract was suspected after cytotoxicity and phosphatase 2A inhibition testing. It was confirmed by ADAM derivatization, immunoaffinity extraction and liquid chromatography with fluorimetric detection analysis as well as by liquid microchromatography with mass spectrometric detection. Results indicate that the P. arenarium strain was toxinogenic and could be potentially involved in the toxin production associated with the human diseases, diarrhetic shellfish poisoning and possibly ciguatera fish poisoning in the SW Indian Ocean area.

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).

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.

[Anesthesia in the HIV positive or AIDS patient]. / Anesthésie du patient VIH positif ou atteint du SIDA.

OBJECTIVE: To analyze the current anaesthetic management of HIV/AIDS patients. DATA SOURCES: References were obtained from computerized bibliographic research (Medline), recent review articles, the library of the service, personal files. STUDY SELECTION: Original articles, reviews, cases reports, letters to the editor in French and English were analyzed and selected. DATA EXTRACTION: Current data on HIV infection, perioperative clinical and biological symptoms, arguments for choice of the type of anaesthesia, risks of transmitting HIV to health care workers and protective measures were extracted. DATA SYNTHESIS: Twenty per cent of HIV-positive patients require surgery during their illness. Anaesthesia and surgery decrease cell mediated immunity and modify the activity of immune mediators. These changes are more pronounced under general anaesthesia compared to regional anaesthesia. They are transient and not clinically significant. Poor information is available concerning the perioperative management of HIV-positive patients and the effects of anesthesia on their immune status. Preoperative evaluation focuses on the following three important data: patient's status, surgery, and anaesthesia. In patients in good clinical conditions who comply with treatment, the anesthetist assesses the effects of the antiretroviral treatment and the risk of interactions between anaesthetic and antiretroviral agents. Etomidate, atracurium, cisatracurium, remifentanil and desflurane are not dependent on hepatic metabolism by the cytochrome P450 system. In patients in bad clinical conditions or in patients who do not comply with treatment, attention focuses on cardiovascular, pulmonary, neurologic and nutritional status. The specific antiretroviral treatment is not discontinued in the perioperative period, as far as compatible with the type of surgery and associated dysfunction of the digestive tract. Regional anaesthesia offers the benefits of not interfering with the immune system and antiretroviral agents. However, the viral infection can be enhanced by regional anaesthesia due to the cofactors' effect of local anaesthetic agents in cerebrospinal fluid. Homologous blood transfusion is not recommended as it increases postoperative infection and viral activation. Erythropoietin can be of benefit in selected cases. In the future, supportive immunotherapy will probably be the main tool for perioperative management of HIV and AIDS patients.

[Nosocomial meningitis in the adult]. / Méningites nosocomiales de l'adulte.

Cerebrospinal fluid (CSF) has anti-infectious defense abilities similar to those of the serum of a neutropenic patient. A septic inoculation as in case of nosocomial meningitis (NM), results rapidly in microbial proliferation with major alterations to the blood brain barrier, cerebral oedema and loss of autoregulation of the cerebral blood flow. Arterial hypotension during NM may induce focal or global cerebral ischaemia. The incidence of NM is increasing, staphylococci and Gram negative bacilli being the most frequent pathological agents. Clinical symptoms are not specific in this postoperative and post-traumatic context. In this context, the analysis of pleocytosis and the increased protein content of CSF is disturbing. Numerous clinical conditions may cause NM. Among them, NM from spinal puncture is an important issue for anaesthetists, while combined epidural and spinal anaesthesia carry the highest risks. Cutaneous contamination plays a major role. Half of the post-operative infections after neurosurgery are due to NM, and CSF leakage, iterative operations and surgery in contaminated conditions are the main risk factors. Antibioprophylaxis for postoperative NM is validated for clean and clean-contaminated surgery. Some consider that only procedures of more than two hours require this prophylaxis. Prophylaxis is targeted on staphylococci. Other preventive measures (drainage of less than 24 h, head shaving and prevention of CSF leakage) are of major importance. Antibiotherapy should be guided by the same considerations as for community acquired meningitis, associated with specific issues in the surgical context (presence of foreign material and CSF blockage).

Multiresidue analysis of pesticides using new laminar extraction disks and liquid chromatography and application to the French priority list.

The national French lists of priority pesticides to be monitored in drinking and/or surface water contain various pesticides with a wide range of physico-chemical properties and can be modified on a regional scale with the addition of other pesticides, depending on local agricultural usage. A multiresidue extraction method is presented using new disk formats engineered for high throughput that are particularly well adapted to the extraction of compounds from high volumes of dirty samples. However, because of the occurrence of very polar and very apolar compounds in the lists, two procedures are required for the extraction step. Divinylbenzene disks were used to extract the more polar compounds, as well as the moderately polar or acidic ones. For apolar pesticides, a C18 silica disk was used, and 10% methanol was added to the water sample before percolation in order to avoid adsorption on the flasks and connecting tubes. Since 1 l of surface water is extracted in less than 5 min without previous filtration using these new laminar disks, the percolation time is no longer a limiting factor in the analysis scheme of surface water samples containing suspended matter. The sample volume can be easily increased in order to reach lower detection limits, provided that the extraction conditions have been optimized in order to minimize the amount of co-extracted and interfering substances. A considerable decrease in the effect of the humic and fulvic substances was achieved using divinylbenzene disks, which allows the samples to be handled at pH 6, for the polar pesticides. Moreover, the necessary addition of 10% methanol in the samples for the extraction of the apolar ones also considerably decreases the amount of co-extracted analytes. The time of the whole sample preparation sequence, i.e., conditioning of the disks, sample extraction, sample clean-up and desorption, is achieved within 10 min for six simultaneous samples. Detection limits in the range 0.01-0.05 microgram l-1 are easily obtained for most pesticides contained in the national lists from 1 l of real surface water samples. The two procedures described in this work allow the handling of any compounds having a water-octanol constant, log Kow, in the range 1-6.