Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca2+] ([Ca2+]i) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca2+]i were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.

Secondary transfer of organic gunshot residues: Empirical data to assist the evaluation of three scenarios.

The present study aimed at providing data to assess the secondary transfer of organic gunshot residues (OGSR). Three scenarios were evaluated in controlled conditions, namely displacing a firearm from point A to point B, a simple handshake and an arrest involving handcuffing on the ground. Specimens were collected from the firearm, the hands of the shooter and the non-shooter undergoing the secondary transfer in order to compare the amounts detected. Secondary transfer was observed for the three scenarios, but to a different extent. It was found that displacing a firearm resulted in secondary transfer in <50% of the experiments. The firearm also had an influence, as contrary to the pistol, no secondary OGSR were detected using the revolver. Shaking the hand of the shooter also transferred OGSR to the non-shooter's hand. In that case, the amount of OGSR was generally higher on the shooter than on the non-shooter. Finally, the largest secondary transfer was observed after the arrest with handcuffing with positive results in all cases using the pistol. In that scenario, the amounts on the shooter and the non-shooter were in the same range. This study highlights that the secondary transfer must be taken into account in the interpretation of OGSR. Indeed, an individual's hands might be contaminated by handling a firearm or having physical contact with a shooter.

Validation of the Mass-Extraction-Window for Quantitative Methods Using Liquid Chromatography High Resolution Mass Spectrometry.

A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.

Static coatings for the analysis of intact monoclonal antibody drugs by capillary zone electrophoresis.

This work reports on the investigation of the optimal conditions associated with good isoform resolution, minimized adsorption, and acceptable repeatability for the analysis of intact monoclonal antibodies. To this end, various static capillary coatings were evaluated, and major trends were observed. For a positively charged coating, a low EOF mobility value was the main criterion for satisfactory resolution. For neutral coatings, adsorption was the main concern. If the coating does not fully cover the surface silanols, electrostatic interactions tend to decrease protein recovery through irreversible adsorption. The influence of the BGE pH was also investigated, as the resolution of monoclonal antibody isoforms is driven by their charge difference. The nature and concentration of the BGE were proven to be important, as an ampholytic compound such as ε-aminocaproic acid was found to decrease adsorption compared with ammonium acetate. With the optimal conditions determined during method development, a complete investigation according to four criteria, i.e. migration time, separation efficiency, EOF mobility, and protein recovery, did not present evidence of any adsorption. Finally, the repeatability and intermediate precision were assessed, and low variability was demonstrated with an RSD less than 1.3% for the main peak migration time.

Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS.

Two major milk whey proteins, ß-lactoglobulin and α-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for ß-lactoglobulin and α-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for ß-lactoglobulin and α-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk.

Total serum IgE quantification by microfluidic ELISA using magnetic beads.

The present work reports on the quantification of total IgE in human serum using a microanalytical device whose fluidics is driven by gravity and capillary forces only. Thanks to the eight parallel microchannels in each microchip, calibration and sample analysis are performed simultaneously. A mixture of magnetic bead/analyte/second antibody is incubated off-line and then percolated through the channels where magnetic beads are trapped, enabling the separation of the solid phase from the excess reagents. The entire assay is performed in less than 1 h, and thanks to the miniaturized format, only a small volume of serum is required. Non-specific adsorption was first investigated and a blocking agent compatible with this allergy-based test was chosen. Then, the assay was optimized by determining the best magnetic bead and labelled antibody concentrations. After achievement of a calibration curve with a reference material, the protocol was applied to total IgE quantification of a patient serum sample that showed results in good accordance with those obtained by ImmunoCap® and Immunoaffinity capillary electrophoresis measurements. A detection limit of 17.5 ng ml(-1) was achieved and good reproducibility (RSD < 10%) inter- and intra-chip was observed.

An application example of the likelihood ratio approach to the evaluation of organic gunshot residues using a fictional scenario and recently published data.

The analysis of gunshot residues (GSR) can provide important information with regard to the involvement of a person of interest (POI) in a firearm-related incident. Organic gunshot residues (OGSR) have been investigated in order to provide additional and complementary information to the traditional inorganic gunshot residue (IGSR) particles detected by scanning electron microscopy (SEM). Currently, many procedures and analytical methods have been developed to detect OGSR-related compounds collected from the shooter's hands. However, such studies provide no information regarding the inclusion of such results in an activity level evaluation for discharging a firearm. The aim of this article is to assess the feasibility of using the likelihood ratio (LR) approach as a tool to evaluate OGSR results for activity level propositions. The developed model focuses on the assignment of an LR for several compounds detected in OGSR. A simple worst-case simulation was investigated in order to assess the applicability of the LR approach to evaluate OGSR traces. This simulation highlighted the importance of addressing an appropriate pair of activity level propositions when evaluating the results.

Bubble cell for magnetic bead trapping in capillary electrophoresis.

A bubble cell capillary classically used to extend the optical path length for UV-vis detection is employed here to trap magnetic beads. With this system, a large amount of beads can be captured without inducing a strong pressure drop, as it is the case with magnetic beads trapped in a standard capillary, thereby having less effect on the experimental conditions. Using numerical simulations and microscopic visualizations, the capture of beads inside a bubble cell was investigated with two magnet configurations. Pressure-driven and electro-osmotic flow velocities were measured for different amounts of protein-A-coated beads or C18-functionalized beads (RPC-18). Solid-phase extraction of a model antibody on protein-A beads and preconcentration of fluorescein on RPC-18 beads were performed as proof of concept experiments.

LEPA: from proteomics to energy conversion.

This review article summarizes the different fields of research at the Laboratoire d'Electrochimie Physique et Analytique at the Ecole Polytechnique Fédérale de Lausanne. The research areas covered include charge transfer reactions at soft interfaces, bio-analytical microchips and electrophoretic methods, electrochemical ionization methods for mass spectrometry and Scanning Electrochemical Microscopy (SECM).

Use of ion mobility-high resolution mass spectrometry in metabolomics studies to provide near MS/MS quality data in a single injection.

The use of ion mobility separations (IMSs) in metabolomics approaches has started to be deeply explored in the last years. In this work, the use of liquid chromatography (LC) coupled to IMS-quadrupole time-of-flight mass spectrometry (QTOF MS) has been evaluated in a metabolomics experiments using single injection of the samples. IMS has allowed obtaining cleaner fragmentation spectra, of nearly tandem MS quality, in data-independent acquisition mode. This is much useful in this research area as a second injection, generally applied in LC-QTOF MS workflows to obtain tandem mass spectra, is not necessary, saving time and evading possible compound degradation. As a case study, the smoke produced after combustion of herbal blends used to spray synthetic cannabinoids has been selected as study matrix. The smoke components were trapped in carbon cartridges, desorbed and analyzed by LC-IMS-QTOF MS using different separation mechanisms (reversed phase and HILIC) and acquiring in both positive and negative mode to widen the chemical domain. Partial Least Squares-Discriminant Analysis highlighted several compounds, and ratio between N-Isopropyl-3-(isoquinolinyl)-2-propen-1-amine and quinoline allowed differentiating between tobacco and herbal products. These two compounds were tentatively identified using the cleaner fragmentation spectra from a single injection in the IMS-QTOF MS, with additional confidence obtained by retention time (Rt) and collisional cross section (CCS) prediction using artificial neural networks. Data from this work show that LC-IMS-QTOF is an efficient technique in untargeted metabolomics, avoiding re-injection of the samples for elucidation purposes. In addition, the prediction models for Rt and CCS resulted of help in the elucidation process of potential biomarkers.

Prevalence of organic gunshot residues in police vehicles.

The present study investigated the organic gunshot residue (OGSR) background level of police vehicles in Switzerland. Specimens from 64 vehicles belonging to two regional police services were collected and analysed by LC-MS in positive mode. The driver's and back seats were sampled separately to monitor potential differences between locations and to assess the risks of a suspect being contaminated by OGSR during transportation to a police station. The results showed that most of the 64 vehicles were uncontaminated (44 driver's seats and 38 back seats respectively). Up to six of the seven targeted compounds were detected in a single sample, once on a driver's seat and twice on back seats. The contamination frequency generally decreased as the number of compounds detected together increased. The amounts detected were in the low ng range and less than amounts generally detected just after discharge on a shooter. Our data indicated that detecting a combination of four or more compounds on a police vehicle seat appears to be a relatively rare occurrence. The background contamination observed was most probably due to secondary transfer from police officers (e.g. through recent participation in a shooting session or firearm manipulation) or from firearms stored in the vehicles. The present results might be used as a recommendation to minimize contact of a suspect with contaminated surfaces if OGSR is implemented in routine work in parallel to IGSR analysis.

Comparison of three collection methods for the sodium rhodizonate detection of gunshot residues on hands.

The aim of this study was to compare three gunshot residue (GSR) collection methods used in conjunction with chemographic detection applied by different regional Swiss police services. The specimens were collected from the hands of a shooter with either filter paper (Filter method) or adhesive foil. The adhesive foil was then either applied against photographic paper during visualisation (AF Photo method) or coated with a layer of polyvinyl alcohol (AF PVAL method). The experiments involved two conditions of the examined hands, i.e. dry and humidified. The residues were revealed using the sodium rhodizonate test (SRT). Preliminary tests assessing the possibility of conducting a confirmatory Scanning Electron Microscopy coupled to Energy Dispersive X-ray spectroscopy (SEM/EDX) analysis after the chemographic test were performed on a number of specimens by cutting positive spots and mounting them on stubs. Obtained results were compared in terms of effectiveness - number of positive spots, time requirements, quality of subsequent SEM-EDX analysis, ease of use and cost. The Filter method generally yielded a high-quality detection with both dry and humidified hands, as well as a simple, quick and efficient confirmation by SEM/EDX. The AF Photo performed well on dry hands, but not on humidified hands. The AF PVAL method performance was lower compared to the other methods in both examined conditions of the hands. The SEM/EDX analysis showed that the Filter and AF PVAL method provided satisfactory results when a sufficient carbon coating thickness was applied to the cuttings. It was also observed that the thinner the PVAL layer, the better the quality of the spectra and obtained images in SEM/EDX. Furthermore, the surface of the photographic paper did not seem to be conductive, even after the application of a thick layer of carbon. In conclusion, the Filter method gave the best overall results, but its application required slightly more time and expertise than the two other methods.

Combined Collection and Analysis of Inorganic and Organic Gunshot Residues.

Gunshot residue (GSR) analysis and their interpretation provide crucial information on a criminal investigation involving the use of firearms. To date, several approaches have been proposed for the implementation of a combined sampling and analysis of inorganic (IGSR) and organic GSR (OGSR). However, it is not clear at this stage if concurrent analyses of both types of residue might be detrimental to the analysis of IGSR currently applied in forensic laboratories. Thus, this work aims to compare and evaluate three different protocols for the combined collection and analysis of IGSR and OGSR. These methods, respectively, involve the use of a modified stub (with two halves, one for the detection of IGSR and the other for the analysis of OGSR); the sequential recovery of GSR with two stubs mounted with different adhesives (double-sided carbon tape and Tesa® TACK) and the sequential analysis of IGSR and OGSR from a single carbon stub following carbon deposition. The detection of IGSR was carried out using SEM-EDX, while OGSR analysis was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Obtained results for experiments performed using Geco Sinoxid® ammunition indicated that sequential analysis was the most suitable protocol for the combined collection and analysis of both IGSR and OGSR. A higher number of inorganic (characteristic and consistent) particles and higher concentrations of ethylcentralite, N-nitrosodiphenylamine, diphenylamine, and nitroglycerin were recovered with this method.

Magnetic forces produced by rectangular permanent magnets in static microsystems.

Finite element numerical simulations were carried out in 2D geometries to map the magnetic field and force distribution produced by rectangular permanent magnets as a function of their size and position with respect to a microchannel. A single magnet, two magnets placed in attraction and in repulsion have been considered. The goal of this work is to show where magnetic beads are preferentially captured in a microchannel. These simulations were qualitatively corroborated, in one geometrical case, by microscopic visualizations of magnetic bead plug formation in a capillary. The results show that the number of plugs is configuration dependent with: in attraction, one plug in the middle of the magnets; in repulsion, two plugs near the edges of the magnets; and with a single magnet, a plug close to the center of the magnet. The geometry of the magnets (h and l are the height and length of the magnets respectively) and their relative spacing s has a significant impact on the magnetic flux density. Its value inside a magnet increases with the h/l ratio. Consequently, bar magnets produce larger and more uniform values than flat magnets. The l/s ratio also influences the magnetic force value in the microchannel, both increasing concomitantly for all the configurations. In addition, a zero force zone in the middle appears in the attraction configuration as the l/s ratio increases, while with a single magnet, the number of maxima and minima goes from one to two, producing two focusing zones instead of only one.

Soft stylus probes for scanning electrochemical microscopy.

A soft stylus microelectrode probe has been developed to carry out scanning electrochemical microscopy (SECM) of rough, tilted, and large substrates in contact mode. It is fabricated by first ablating a microchannel in a polyethylene terephthalate thin film and filling it with a conductive carbon ink. After curing the carbon track and lamination with a polymer film, the V-shaped stylus was cut thereby forming a probe, with the cross section of the carbon track at the tip being exposed either by UV-photoablation machining or by blade cutting followed by polishing to produce a crescent moon-shaped carbon microelectrode. The probe properties have been assessed by cyclic voltammetry, approach curves, and line scans over electrochemically active and inactive substrates of different roughness. The influence of probe bending on contact mode imaging was then characterized using simple patterns. Boundary element method simulations were employed to rationalize the distance-dependent electrochemical response of the soft stylus probes.

Magnetic track array for efficient bead capture in microchannels.

Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length (L (m) = 2, 5, 10 mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnets.

Surveys of organic gunshot residue prevalence: Comparison between civilian and police populations.

This study investigated the prevalence of eight OGSR compounds in a civilian and a police population. Specimens were collected from the hands and sleeves of 122 civilians and 115 individuals working in police services using carbon stubs. Data was acquired using liquid chromatography coupled to tandem mass spectrometry. Results indicated a non-negligible prevalence in the civilian sample, with 18% of the 122 civilians sampled having one or more OGSR related compounds on their hands and 11.5% on their wrists or sleeves. For the police population, the prevalence was logically higher than for civilians, with 36.5% of the hand specimens and 33% of the wrist specimens positive for one or more compounds. A higher prevalence was expected for the second population due to the possession of service weapons regularly used during shooting exercises. These results demonstrate that the presence of one OGSR compound is not a rare occurrence, even in a civilian population. Considering the results of this research together with information on alternative sources of the targeted OGSR compounds can serve as a basis for OGSR interpretation in casework.

An investigation on the secondary transfer of organic gunshot residues.

Gunshot residues (GSR) are an important forensic trace in firearm-related events. Currently, routine GSR analyses focus on the detection and characterisation of the inorganic components (IGSR). The increasing prevalence of heavy metal-free ammunition challenges these current protocols and there is an increasing interest in how the organic components of GSR (OGSR) can provide complementary information. Similar to the situation with IGSR, OGSR compounds originally deposited on the shooter during the firing process may further be transferred onto another individual or surface. Hence, the aim of this study was to provide additional information regarding the risk of a secondary transfer of OGSR. Two scenarios were investigated, the first one related to the arrest process and the possibilities of a secondary transfer arising between a shooter onto a non-shooter (e.g. between a police officer and a person of interest (POI)). The second scenario concerned the transfer of OGSR onto the non-shooter after handling a firearm for few minutes without discharging it. One calibre was chosen, the .40 S&W calibre, used by several Australian State police forces. A secondary transfer was observed in all cases for the two scenarios investigated, for three compounds of interest: ethylcentralite (EC), diphenylamine (DPA), N-nitrosodiphenylamine (N-nDPA). The firearm handling scenario resulted in a larger secondary transfer to that of the arrest scenario. Overall, the amounts of OGSR detected on the non-shooter were generally lower than that detected on the shooter and controls after the arrest scenario. The results of this study provide complementary knowledge about OGSR, which can be further used to improve the current practice and the interpretation of OGSR evidence. In particular, it highlights that the secondary transfer proposition must be considered during the interpretation of forensic findings, especially when small amounts of OGSR target compounds are detected.

Remarkable tuning of the photophysical properties of bifunctional lanthanide tris(dipicolinates) and its consequence on the design of bioprobes.

Derivatives of dipicolinic acid with a polyoxyethylene pendant arm at the pyridine 4-position have been functionalized for potential grafting with biological material. Four ligands with different terminal functions (alcohol, methoxy, phtalimide and amine) have been synthesized, which react with trivalent lanthanide ions Ln (III) to yield triple helical [Ln(L) 3] (3-) complexes, as shown by NMR and UV-vis titrations. The tris chelates display large thermodynamic stability with log beta 13 approximately 19-20 for all Eu (III) complexes for instance. Photophysical measurements reveal adequate sensitization of the metal-centered luminescence in the europium (eta sens = 33-72%) and terbium complexes, which is modulated by the nature of the terminal function. The lifetimes of the metal-centered excited states are long, up to 1.4 ms for [Eu(L) 3] (3-) and 1.6 ms for [Tb(L) 3] (3-) at room temperature, in line with hydration numbers essentially equal to zero. Quantum yields are as high as 29% for the [Eu( L ( NH2 )) 3] (3-) and 18% for the [Tb( L ( OH )) 3] (3-) tris chelates in water at physiological pH. These series of complexes demonstrate the extent of fine-tuning achievable for lanthanide luminescent probes and are simple models for investigating the effect of binding to biological molecules on the metal-centered luminescent properties.

A forensic investigation on the persistence of organic gunshot residues.

Gunshot residues (GSR) are a potential form of forensic traces in firearm-related events. In most forensic laboratories, GSR analyses focus on the detection and characterisation of the inorganic components (IGSR), which are mainly particles containing mixtures of lead, barium and antimony originating from the primer. The increasing prevalence of heavy metal-free ammunition challenges the current protocols used for IGSR analysis. To provide complementary information to IGSR particles, the current study concentrated on the organic components (OGSR) arising from the combustion of the propellant. The study focused on four compounds well-known as being part of OGSR: ethylcentralite (EC), methylcentralite (MC), diphenylamine (DPA), N-nitrosodiphenylamine (N-nDPA). This study assessed the retention of these OGSR traces on a shooter's hands. The overall project aim was to provide appropriate information regarding OGSR persistence, which can be suitable to be integrated into the interpretation framework of OGSR as recommended by the recent ENFSI Guideline for Evaluative Reporting in Forensic Science. The persistence was studied through several intervals ranging from immediately after discharge to four hours and two ammunition calibres were chosen: .40 S&W calibre, used by the NSW Police Force; and .357 Magnum, which is frequently encountered in Australian casework. This study successfully detected the compounds of interest up to four hours after discharge. The trends displayed a large decrease in the amount detected during the first hour. A large variability was also observed due to numerous factors involved in the production, deposition and collection of OGSR.