Water mobility and microstructure of gluten network during dough mixing using TD NMR.

This work aimed to establish the relationships between flour components, dough behaviour and changes in water distribution at mixing. TD NMR was used to track water distribution in dough during mixing for different mixing times and hydration levels. Four commercial wheat flours with distinct characteristics were expressly selected to exhibit various dough behaviours at mixing. TD NMR measurements of mixed dough samples revealed four to five water mobility domains depending on the flour type and the mixing modality. A classification tree procedure was used to identify characteristic patterns of water mobility in dough, called hydration states (HS). The HS changes with experimental conditions are highly dependent on flour characteristics, and HS were assigned to physical/chemical changes in the gluten network during dough formation. This study proposes an interpretation of the water distribution in dough based on gluten network development. This will help to adapt the mixing process to the flour characteristics.

Usability Testing and Technology Acceptance of an mHealth App at the Point of Care During Simulated Pediatric In- and Out-of-Hospital Cardiopulmonary Resuscitations: Study Nested Within 2 Multicenter Randomized Controlled Trials.

BACKGROUND: Mobile apps are increasingly being used in various domains of medicine. Few are evidence-based, and their benefits can only be achieved if end users intend to adopt and use them. To date, only a small fraction of mobile apps have published data on their field usability and end user acceptance results, especially in emergency medicine. OBJECTIVE: This study aims to determine the usability and acceptance of an evidence-based mobile app while safely preparing emergency drugs at the point of care during pediatric in- and out-of-hospital cardiopulmonary resuscitations by frontline caregivers. METHODS: In 2 multicenter randomized controlled parent trials conducted at 6 pediatric emergency departments from March 1 to December 31, 2017, and 14 emergency medical services from September 3, 2019, to January 21, 2020, the usability and technology acceptance of the PedAMINES (Pediatric Accurate Medication in Emergency Situations) app were evaluated among skilled pediatric emergency nurses and advanced paramedics when preparing continuous infusions of vasoactive drugs and direct intravenous emergency drugs at pediatric dosages during standardized, simulation-based, pediatric in- and out-of-hospital cardiac arrest scenarios, respectively. Usability was measured using the 10-item System Usability Scale. A 26-item technology acceptance self-administered survey (5-point Likert-type scales), adapted from the Unified Theory of Acceptance and Use of Technology model, was used to measure app acceptance and intention to use. RESULTS: All 100% (128/128) of nurses (crossover trial) and 49.3% (74/150) of paramedics (parallel trial) were assigned to the mobile app. Mean total scores on the System Usability Scale were excellent and reached 89.5 (SD 8.8; 95% CI 88.0-91.1) for nurses and 89.7 (SD 8.7; 95% CI 87.7-91.7) for paramedics. Acceptance of the technology was very good and rated on average >4.5/5 for 5 of the 8 independent constructs evaluated. Only the image construct scored between 3.2 and 3.5 by both participant populations. CONCLUSIONS: The results provide evidence that dedicated mobile apps can be easy to use and highly accepted at the point of care during in- and out-of-hospital cardiopulmonary resuscitations by frontline emergency caregivers. These findings can contribute to the implementation and valorization of studies aimed at evaluating the usability and acceptance of mobile apps in the field by caregivers, even in critical situations. TRIAL REGISTRATION: ClinicalTrials.gov NCT03021122; https://clinicaltrials.gov/ct2/show/NCT03021122. ClinicalTrials.gov NCT03921346; https://clinicaltrials.gov/ct2/show/NCT03921346. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s13063-019-3726-4.

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.

Impact of a Mobile App on Paramedics' Perceived and Physiologic Stress Response During Simulated Prehospital Pediatric Cardiopulmonary Resuscitation: Study Nested Within a Multicenter Randomized Controlled Trial.

BACKGROUND: Out-of-hospital cardiac arrests (OHCAs) are stressful, high-stake events that are associated with low survival rates. Acute stress experienced in this situation is associated with lower cardiopulmonary resuscitation performance in calculating drug dosages by emergency medical services. Children are particularly vulnerable to such errors. To date, no app has been validated to specifically support emergency drug preparation by paramedics through reducing the stress level of this procedure and medication errors. OBJECTIVE: This study aims to determine the effectiveness of an evidence-based mobile app compared with that of the conventional preparation methods in reducing acute stress in paramedics at the psychological and physiological levels while safely preparing emergency drugs during simulated pediatric OHCA scenarios. METHODS: In a parent, multicenter, randomized controlled trial of 14 emergency medical services, perceived and physiologic stress of advanced paramedics with drug preparation autonomy was assessed during a 20-minute, standardized, fully video-recorded, and highly realistic pediatric OHCA scenario in an 18-month-old child. The primary outcome was participants' self-reported psychological stress perceived during sequential preparations of 4 intravenous emergency drugs (epinephrine, midazolam, 10% dextrose, and sodium bicarbonate) with the support of the PedAMINES (Pediatric Accurate Medication in Emergency Situations) app designed to help pediatric drug preparation (intervention) or conventional methods (control). The State-Trait Anxiety Inventory and Visual Analog Scale questionnaires were used to measure perceived stress. The secondary outcome was physiologic stress, measured by a single continuous measurement of the participants' heart rate with optical photoplethysmography. RESULTS: From September 3, 2019, to January 21, 2020, 150 advanced paramedics underwent randomization. A total of 74 participants were assigned to the mobile app (intervention group), and 76 did not use the app (control group). A total of 600 drug doses were prepared. Higher State-Trait Anxiety Inventory-perceived stress increase from baseline was observed during the scenario using the conventional methods (mean 35.4, SD 8.2 to mean 49.8, SD 13.2; a 41.3%, 35.0 increase) than when using the app (mean 36.1, SD 8.1 to mean 39.0, SD 8.4; a 12.3%, 29.0 increase). This revealed a 30.1% (95% CI 20.5%-39.8%; P<.001) lower relative change in stress response in participants who used the app. On the Visual Analog Scale questionnaire, participants in the control group reported a higher increase in stress at the peak of the scenario (mean 7.1, SD 1.8 vs mean 6.4, SD 1.9; difference: -0.8, 95% CI -1.3 to -0.2; P=.005). Increase in heart rate during the scenario and over the 4 drugs was not different between the 2 groups. CONCLUSIONS: Compared with the conventional method, dedicated mobile apps can reduce acute perceived stress during the preparation of emergency drugs in the prehospital setting during critical situations. These findings can help advance the development and evaluation of mobile apps for OHCA management and should be encouraged. TRIAL REGISTRATION: ClinicalTrials.gov NCT03921346; https://clinicaltrials.gov/ct2/show/NCT03921346. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1186/s13063-019-3726-4.

Effect of a Mobile App on Prehospital Medication Errors During Simulated Pediatric Resuscitation: A Randomized Clinical Trial.

Importance: Medication errors are a leading cause of injury and avoidable harm, affecting millions of people worldwide each year. Children are particularly susceptible to medication errors, but innovative interventions for the prevention of these errors in prehospital emergency care are lacking. Objective: To assess the efficacy of an evidence-based mobile app in reducing the occurrence of medication errors compared with conventional preparation methods during simulated pediatric out-of-hospital cardiac arrest scenarios. Design, Setting, and Participants: This nationwide, open-label, multicenter, randomized clinical trial was conducted at 14 emergency medical services centers in Switzerland from September 3, 2019, to January 21, 2020. The participants were 150 advanced paramedics with drug preparation autonomy. Each participant was exposed to a 20-minute, standardized, fully video-recorded, realistic pediatric out-of-hospital cardiac arrest cardiopulmonary resuscitation scenario concerning an 18-month-old child. Participants were tested on sequential preparations of 4 intravenous emergency drugs of varying degrees of preparation difficulty (epinephrine, midazolam, 10% dextrose, and sodium bicarbonate). Intervention: Participants were randomized (1:1 ratio) to the support of an app designed to assist with pediatric drug preparation (intervention; n = 74) or to follow conventional drug preparation methods without assistance (control; n = 76). Main Outcomes and Measures: The primary outcome was the rate of medication errors, defined as a failure in drug preparation according to predefined, expert consensus-based criteria. Logistic regression models with mixed effects were used to assess the effect of the app on binary outcomes. Secondary outcomes included times to drug preparation and delivery, assessed with linear regression models with mixed effects. Results: In total, 150 advanced paramedics (mean [SD] age, 35.6 [7.2] years; 101 men [67.3%]; mean [SD] time since paramedic certification, 8.0 [6.2] years) participated in the study and completed 600 drug preparations. Of 304 preparations delivered using the conventional method, 191 (62.8%; 95% CI, 57.1%-68.3%) were associated with medication errors compared with 17 of 296 preparations delivered using the app (5.7%; 95% CI, 3.4%-9.0%). When accounting for repeated measures, with the app, the proportion of medication errors decreased in absolute terms by 66.5% (95% CI, 32.6%-83.8%; P < .001), the mean time to drug preparation decreased by 40 seconds (95% CI, 23-57 seconds; P < .001), and the mean time to drug delivery decreased by 47 seconds (95% CI, 27-66 seconds; P < .001). The risk of medication errors varied across drugs with conventional methods (19.7%-100%) when compared with the app (4.1%-6.8%). Conclusions and Relevance: Compared with conventional methods, the use of a mobile app significantly decreased the rate of medication errors and time to drug delivery for emergency drug preparation in a prehospital setting. Dedicated mobile apps have the potential to improve medication safety and change practices in pediatric emergency medicine. Trial Registration: ClinicalTrials.gov Identifier: NCT03921346.

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.

Enterovirus, parechovirus, adenovirus and herpes virus type 6 viraemia in fever without source.

OBJECTIVES: To evaluate the potential associations between fever without a source (FWS) in children and detection of human enterovirus (HEV), human parechovirus (HPeV), adenovirus (AdV) and human herpesvirus type 6 (HHV-6) in the plasma; and to assess whether the detection of viruses in the plasma is associated with a reduced risk of serious bacterial infection (SBI) and antibiotic use. DESIGN AND SETTING: Between November 2015 and December 2017, this prospective, single-centre, diagnostic study tested the plasma of children <3 years old with FWS. Real-time (reverse-transcription) PCR for HEV, HPeV, AdV and HHV-6 was used in addition to the standardised institutional work-up. A control cohort was also tested for the presence of viruses in their blood. RESULTS: HEV, HPeV, AdV and HHV-6 were tested for in the plasma of 135 patients of median age 2.4 months old. At least one virus was detected in 47 of 135 (34.8%): HEV in 14.1%, HHV-6 in 11.1%, HPeV in 5.9% and AdV in 5.2%. There was no difference in antibiotic use between patients with or without virus detected, despite a relative risk of 0.2 for an SBI among patients with viraemia. Controls were less frequently viraemic than children with FWS (6.0% vs 34.8%; p<0.001). CONCLUSIONS: HEV, HPeV, AdV and HHV-6 are frequently detected in the plasma of children with FWS. Antibiotic use was similar between viraemic and non-viraemic patients despite a lower risk of SBI among patients with viraemia. Point-of-care viral PCR testing of plasma might reduce antibiotic use and possibly investigations and admission rates in patients with FWS. TRIAL REGISTRATION NUMBER: NCT03224026.

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).

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.

A mobile device app to reduce prehospital medication errors and time to drug preparation and delivery by emergency medical services during simulated pediatric cardiopulmonary resuscitation: study protocol of a multicenter, prospective, randomized controlled trial.

BACKGROUND: Emergency drug preparation and administration in children is both complex and time-consuming and places this population at a higher risk than adults for medication errors. Moreover, survival and a favorable neurological outcome from cardiopulmonary resuscitation are inversely correlated to drug preparation time. We developed a mobile device application (the pediatric Accurate Medication IN Emergency Situations (PedAMINES) app) as a step-by-step guide for the preparation to delivery of drugs requiring intravenous injection. In a previous multicenter randomized trial, we reported the ability of this app to significantly reduce in-hospital continuous infusion medication error rates and drug preparation time compared to conventional preparation methods during simulation-based pediatric resuscitations. This trial aims to evaluate the effectiveness of this app during pediatric out-of-hospital cardiopulmonary resuscitation. METHODS/DESIGN: We will conduct a multicenter, prospective, randomized controlled trial to compare the PedAMINES app with conventional calculation methods for the preparation of direct intravenously administered emergency medications during standardized, simulation-based, pediatric out-of-hospital cardiac arrest scenarios using a high-fidelity manikin. One hundred and twenty paramedics will be randomized (1:1) in several emergency medical services located in different regions of Switzerland. Each paramedic will be asked to prepare, sequentially, four intravenously administered emergency medications using either the app or conventional methods. The primary endpoint is the medication error rates. Enrollment will start in mid-2019 and data analysis in late 2019. We anticipate that the intervention will be completed in early 2020 and study results will be submitted in late 2020 for publication (expected in early 2021). DISCUSSION: This clinical trial will assess the impact of an evidence-based mobile device app to reduce the rate of medication errors, time to drug preparation and time to drug delivery during prehospital pediatric resuscitation. As research in this area is scarce, the results generated from this study will be of great importance and may be sufficient to change and improve prehospital pediatric emergency care practice. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03921346. Registered on 18 April 2019.

A mobile device application to reduce medication errors and time to drug delivery during simulated paediatric cardiopulmonary resuscitation: a multicentre, randomised, controlled, crossover trial.

BACKGROUND: Vasoactive drug preparation for continuous infusion in children is both complex and time consuming and places the paediatric population at higher risk than adults for medication errors. We developed a mobile device application (app) as a step-by-step guide for the preparation to delivery of drugs requiring continuous infusion. The app has been previously tested during simulation-based resuscitations in a previous single-centre trial. In this trial, our aim was to assess this app in various hospital settings. METHODS: We did a prospective, multicentre, randomised, controlled, crossover trial to compare this app with an internationally used drug-infusion-rates table for the preparation of continuous drug infusion during standardised, simulation-based, paediatric post-cardiac arrest scenarios using a high-fidelity manikin. The scenarios were split into two study periods to assess the two preparation methods consecutively, separated by a washout distraction manoeuvre. Nurses in six paediatric emergency centres in Switzerland were randomly assigned (1:1) to start the scenario with either the app or the infusion-rates table and then complete the scenario using the other preparation method. The primary endpoint was the proportion of participants committing a medication error, which was defined as a deviation from the correct weight dose of more than 10%, miscalculation of the infusion rate, misprogramming of the infusion pump, or the inability to calculate drug dosage without calculation and guidance help from the study team. The medication error proportions observed with both preparation methods were compared by pooling both study periods, with paired data analysed using the unconditional exact McNemar test for dependent groups with a two-sided α level of 0·05. We did sensitivity analyses to investigate the carryover effect. This trial is registered with ClinicalTrials.gov, number NCT03021122. FINDINGS: From March 1 to Dec 31, 2017, we randomly assigned 128 nurses to start the scenario using the app (n=64) or the infusion-rates table (n=64). Among the 128 drug preparations associated with each of the two methods, 96 (75%, 95% CI 67-82) delivered using the infusion-rates table were associated with medication errors compared with nine (7%, 3-13) delivered using the mobile app. Medication errors were reduced by 68% (95% CI 59-76%; p<0·0001) with the app compared with the table, as was the mean time to drug preparation (difference 148·2 s [95% CI 124·2-172·1], a 45% reduction; p<0·0001) and mean time to drug delivery (168·5 s [146·1-190·8], a 40% reduction; p<0·0001). Hospital size and nurses' experience did not modify the intervention effect. We detected no carryover effect. INTERPRETATION: Critically ill children are particularly vulnerable to medication errors. A mobile app designed to help paediatric drug preparation during resuscitation with the aim to significantly reduce the occurrence of medication errors, drug preparation time, and delivery time could have the potential to change paediatric clinical practice in the area of emergency medicine. FUNDING: Swiss National Science Foundation.

Impact of the lab-score on antibiotic prescription rate in children with fever without source: a randomized controlled trial.

BACKGROUND: The Lab-score, based on the combined determination of procalcitonin, C-reactive protein and urinary dipstick results, has been shown accurate in detecting serious bacterial infections (SBI) in children with fever without source (FWS) on retrospective cohorts. We aimed to prospectively assess the utility of the Lab-score in safely decreasing antibiotic prescriptions in children with FWS and to determine its diagnostic characteristics compared to common SBI biomarkers. METHODS: Randomized controlled trial in children 7 days to 36 months old with FWS, allocated either to the Lab-score group (Lab-score reported, blinded WBC count) or to the control group (WBC, bands and C-reactive protein determined, blinded procalcitonin and Lab-score), followed up until recovery. Demographic data, antibiotic prescription rate, admission rate and diagnostic properties of the Lab-score were analyzed. RESULTS: 271 children were analyzed. No statistically significant difference concerning antibiotic prescription rate was observed: 41.2% (54 of 131) in the Lab-score group and 42.1% (59 of 140) in the control group (p = 1.000). If recommendations based on the Lab-score had been strictly applied, a hypothetical 30.6% treatment rate would have been encountered, compared to the overall 41.7% observed rate (p = 0.0095). A Lab-score ≥3 showed the following characteristics: sensitivity 85.1% (95% CI: 76.5-93.6%), specificity 87.3% (95% CI: 82.7-91.8%), positive predictive value 68.7% (95% CI: 58.7-78.7%), negative predictive value 94.1% (95% CI: 91.5-97.9%), positive and negative likelihood ratios: 6.68 and 0.17 respectively. Area under the receiver operating characteristic curve was best for the Lab-score (0.911, 95% CI: 0.871-0.950). DISCUSSION: No difference regarding antibiotic treatment rate was observed when using the Lab-score, due to lack of adherence to the related recommendations. However, if strictly followed, a significant 26.5% reduction of antibiotic prescriptions would have been encountered. Medical education needs to be reinforced in order to observe rather than treat low-risk well-appearing children with FWS. TRIAL REGISTRATION: ClinicalTrials.gov NCT02179398.

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.

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.

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%.

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.

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.