A preliminary study of rapid-fire high-throughput metabolite analysis using nano-flow injection/Q-TOFMS.

In this study, we demonstrated nano-flow injection analysis (nano-FIA) with quadrupole time-of-flight mass spectrometry (Q-TOFMS) for 17 highly polar intermediates produced during glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). We optimized the analytical conditions for nano-flow injection/Q-TOFMS, and set the flow rate and ion source temperature to 1000 nL/min and 150 °C, respectively. Under optimal conditions, a single run was finished within 3 min, and the RSD value of 50 sequential injections was 4.2%. The method also showed quantitativity of four stable-isotope-labeled compounds (r2 > 0.99), demonstrating its robustness, high repeatability, and specificity. In addition, we compared three sample-preparation methods for rodent blood samples and found that protein precipitation with threefold methanol was the most effective. Finally, we applied the method to plasma samples from the serotonin syndrome (SS) model and control rats, the results of which were evaluated by principal component analysis (PCA). The two groups showed clearly separated PCA score plots, suggesting that the method could successfully catch the differences in metabolic profiles between SS and control rats. The results obtained from our new method were further validated by using the established gas chromatography/tandem mass spectrometry method, which demonstrated that there were good correlations between the two methods (R = 0.902 and 0.958 for lactic acid and malic acid, respectively, each at p < 0.001), thus proving the validity of our method. The method described here enables high-throughput analysis of metabolites and will be of use for the rapid analysis of metabolic profiles. Graphical abstract.

A Decoupled Automation Platform for Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.

Simultaneous and direct determination of urea and creatinine in human urine using a cost-effective flow injection system equipped with in-house contactless conductivity detector and LED colorimeter.

This work presents a cost-effective and simple flow injection analysis (FIA) system for simultaneous and direct determination of urea and creatinine in human urine. The FIA system comprises two in-house detectors, a contactless conductivity detector and a light emitting diode (LED) detector. The contactless detector was built as a flow-through detection cell with axial electrodes, commonly known as capacitively coupled contactless conductivity detector (C4D) and the diode detector was fabricated based on the concept of paired emitter detector diodes (PEDD). With appropriate dilution of urine, the sample is directly injected into a stream of glycine-NaOH buffer pH 8.8 (the gas donor stream) and is carried by the carrier through a urease minicolumn for on-line enzymatic hydrolysis. The generated NH3 diffuses from the carrier stream through a porous polytetrafluoroethylene (PTFE) membrane into a stream of deionized water (the acceptor stream) leading to an increase in the signal at the C4D due to the NH3 dissolution in the water. In this system, creatinine is determined based on the Jaffé reaction by merging a stream of alkaline picrate with the gas donor stream. The change in color is detected using the PEDD equipped with two green LEDs. Under the optimum condition, the linear range of urea and creatinine were 30-240 mg L-1 and 10-500 mg L-1, with limits of detection of 9.0 mg L-1 and 0.9 mg L-1, respectively. The proposed system provides satisfactorily good precision (RSD < 3%), with sample throughput of 31 sample h-1 for the two analytes. The FIA system tolerates potential interference commonly found in human urine. The system was successfully applied and validated with selected reference methods.

Multiple signal amplification chemiluminescence immunoassay for chloramphenicol using functionalized SiO2 nanoparticles as probes and resin beads as carriers.

A novel, rapid and convenient competitive immunoassay for ultrasensitive detection of chloramphenicol residues in shrimp and honey was established combined with flow injection chemiluminescence. The carboxylic resin beads were used as solid phase carriers to load with more coating antigen due to their larger specific surface area and good biocompatibility. The surface of the silica dioxide nanoparticles was modified with aldehyde group to combine with more horseradish peroxidase and the chloramphenicol antibody. There was a competitive process between the chloramphenicol in solution and the immobilized coating antigen to combine with the limited binding site of antibody to form the immunocomplex. Silica dioxide nanoparticles played an important role in enhancing chemiluminescence signal, because the horseradish peroxidase on SiO2 effectively catalyzed the system of luminol-PIP-H2O2. Under optimal conditions, the chemiluminescence intensity decreased linearly with the logarithm of the chloramphenicol concentration in the range of 0.0001 to 100 ng mL-1 and the detection limit (3σ) was 0.033 pg mL-1. This immunosensor demonstrated acceptable stability, high specificity and reproducibility. The horseradish peroxidase-silica dioxide nanoparticle-chloramphenicol antibody complex successfully prepared in this article was firstly applied to the detection of chloramphenicol, and had extremely important meanings for the application of nanoparticles and enzymatic catalysis in the field of chemiluminescence.

Strategies for sample delivery for femtosecond crystallography.

Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

Simultaneous determination of rutin and ascorbic acid in a sequential injection lab-at-valve system.

A green, simple, accurate and highly sensitive sequential injection lab-at-valve procedure has been developed for the simultaneous determination of ascorbic acid (Asc) and rutin using 18-molybdo-2-phosphate Wells-Dawson heteropoly anion (18-MPA). The method is based on the dependence of the reaction rate between 18-MPA and reducing agents on the solution pH. Only Asc is capable of interacting with 18-MPA at pH 4.7, while at pH 7.4 the reaction with both Asc and rutin proceeds simultaneously. In order to improve the precision and sensitivity of the analysis, to minimize reagent consumption and to remove the Schlieren effect, the manifold for the sequential injection analysis was supplemented with external reaction chamber, and the reaction mixture was segmented. By the reduction of 18-MPA with reducing agents one- and two-electron heteropoly blues are formed. The fraction of one-electron heteropoly blue increases at low concentrations of the reducer. Measurement of the absorbance at a wavelength corresponding to the isobestic point allows strictly linear calibration graphs to be obtained. The calibration curves were linear in the concentration ranges of 0.3-24mgL-1 and 0.2-14mgL-1 with detection limits of 0.13mgL-1 and 0.09mgL-1 for rutin and Asc, respectively. The determination of rutin was possible in the presence of up to a 20-fold molar excess of Asc. The method was applied to the determination of Asc and rutin in ascorutin tablets with acceptable accuracy and precision (1-2%).

Ultrasound-assisted dispersive liquid-liquid microextraction of tetracycline drugs from egg supplements before flow injection analysis coupled to a liquid waveguide capillary cell.

A simple, rapid, and efficient ultrasound-assisted dispersive liquid-liquid microextraction (US-DLLME) method was developed for extraction of tetracycline residues from egg supplement samples, with subsequent determination by flow injection analysis (FIA) coupled to a liquid waveguide capillary cell (LWCC) and a controlled temperature heating bath. Tetracyclines react with diazotized p-sulfanilic acid, in a slightly alkaline medium, to form azo compounds that can be measured at 435 nm. The reaction sensitivity improved substantially (5.12-fold) using an in-line heating temperature of 45 °C. Multivariate methodology was used to optimize the factors affecting the extraction efficiency, considering the volumes of extraction and disperser solvents, sonication time, extraction time, and centrifugation time. Good linearity in the range 30-600 µg L(-1) was obtained for all the tetracyclines, with regression coefficients (r) higher than 0.9974. The limits of detection ranged from 6.4 to 11.1 µg L(-1), and the recoveries were in the range 85.7-96.4 %, with relative standard deviation lower than 9.8 %. Analyte recovery was improved by approximately 6 % when the microextraction was assisted by ultrasound. The results obtained with the proposed US-DLLME-FIA method were confirmed by a reference HPLC method and showed that the egg supplement samples analyzed were suitable for human consumption.

Flow-injection chemiluminescence analysis for sensitive determination of atenolol using cadmium sulfide quantum dots.

A sensitive, rapid and simple flow-injection chemiluminescence (CL) system based on the light emitted from KMnO4-cadmium sulfide quantum dots (CdS QDs) reaction in the presence of cetyltrimethylammonium bromide (CTAB) in acidic medium was developed as a CL probe for the sensitive determination of atenolol. Optical and structural features of CdS QDs capped with l-cysteine, which synthesized via hydrothermal approach, were investigated using X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL), and UV-Vis spectroscopy. The CL intensity of KMnO4-CdS QDs-CTAB was remarkably enhanced in the presence of trace level of atenolol. Under optimum experimental conditions, there is a linear relationship between the increase in CL intensity of KMnO4-CdS QDs-CTAB system and atenolol concentration in a range of 0.001 to 4.0 mg L(-1) and 4.0 to 18.0 mg L(-1), with a detection limit (3σ) of 0.0010 mg L(-1). A possible mechanism for KMnO4-CdS QDs-CTAB-atenolol CL reaction is proposed. To prove the practical application of the KMnO4-CdS QDs-CTAB CL method, the method was applied for the determination of atenolol in spiked environmental water samples and commercial pharmaceutical formulation. Furthermore, corona discharge ionization ion mobility spectrometry (CD-IMS) technique was utilized for determination of atenolol.

Analysis of saccharides in beverages by HPLC with direct UV detection.

The present study demonstrates the suitability of direct UV detection for saccharide analysis in HPLC. Under highly alkaline conditions, the non-UV absorbing saccharides are converted by a photo-initiated chemical reaction in the detection cell into malonenolate, which can be detected at 266 nm. A straightforward method for such direct UV detection of saccharides after their separation by anion-exchange chromatography was developed and successfully applied to several beverage samples. Investigation and optimization of the influencing factors using design of experiment resulted in a baseline separation of glucose, fructose, and sucrose within 6 min and LOD values below 0.2 mg L(-1). In addition, a fast, simple and cost-effective flow injection method was developed to estimate the total saccharide concentration. The results of this method applied to beverage samples are in good agreement with the chromatographic method as well as to the saccharide concentration stated by the manufacturer. Finally, a comparison of different commercially available UV detectors and detector cells revealed that sensitive detection requires the use of recently introduced flow cells with extended path length.

Cost-effective flow injection amperometric system with metal nanoparticle loaded carbon nanotube modified screen printed carbon electrode for sensitive determination of hydrogen peroxide.

Various metal nanoparticles (NPs) decorated on carbon nanotube (CNT) was modified on the home-made screen printed carbon electrode (SPCE) in order to enhances sensitivity of hydrogen peroxide (H2O2) determination. The simple casting method was used for the electrode modification. The monometallic and bimetallic NPs modified electrodes were investigated for their electrochemical properties for H2O2 reduction. The Pd-CNT/SPCE is appropriated to measure the H2O2 reduction at a potential of -0.3 V, then this modified electrode was incorporated with a home-made flow through cell and applied in a simple flow injection amperometry (FI-Amp). Some parameters influencing the resulted modified electrode and the FI-Amp system were studied. The proposed detection system was able to detect H2O2 in the range of 0.1-1.0 mM, with detection limit of 20 µM. Relative standard deviation for 100 replicated injections of 0.6 mM H2O2 was 2.3%. The reproducibility of 6 electrodes preparing in 3 different lots was 8.2%. It was demonstrated for determination of H2O2 in disinfectant, hair colorant and milk samples. Recoveries in the range of 90-109% were observed. The developed system provided high stability, good repeatability, high sample throughput and low reagent consumption.

A portable low-cost long-term live-cell imaging platform for biomedical research and education.

Time-resolved visualization and analysis of slow dynamic processes in living cells has revolutionized many aspects of in vitro cellular studies. However, existing technology applied to time-resolved live-cell microscopy is often immobile, costly and requires a high level of skill to use and maintain. These factors limit its utility to field research and educational purposes. The recent availability of rapid prototyping technology makes it possible to quickly and easily engineer purpose-built alternatives to conventional research infrastructure which are low-cost and user-friendly. In this paper we describe the prototype of a fully automated low-cost, portable live-cell imaging system for time-resolved label-free visualization of dynamic processes in living cells. The device is light-weight (3.6 kg), small (22 × 22 × 22 cm) and extremely low-cost (<€1250). We demonstrate its potential for biomedical use by long-term imaging of recombinant HEK293 cells at varying culture conditions and validate its ability to generate time-resolved data of high quality allowing for analysis of time-dependent processes in living cells. While this work focuses on long-term imaging of mammalian cells, the presented technology could also be adapted for use with other biological specimen and provides a general example of rapidly prototyped low-cost biosensor technology for application in life sciences and education.

Automation of 99Tc extraction by LOV prior ICP-MS detection: application to environmental samples.

A new, fast, automated and inexpensive sample pre-treatment method for (99)Tc determination by inductively coupled plasma-mass spectrometry (ICP-MS) detection is presented. The miniaturized approach is based on a lab-on-valve (LOV) system, allowing automatic separation and preconcentration of (99)Tc. Selectivity is provided by the solid phase extraction system used (TEVA resin) which retains selectively pertechnetate ion in diluted nitric acid solution. The proposed system has some advantages such as minimization of sample handling, reduction of reagents volume, improvement of intermediate precision and sample throughput, offering a significant decrease of both time and cost per analysis in comparison to other flow techniques and batch methods. The proposed LOV system has been successfully applied to different samples of environmental interest (water and soil) with satisfactory recoveries, between 94% and 98%. The detection limit (LOD) of the developed method is 0.005 ng. The high durability of the resin and its low amount (32 mg), its good intermediate precision (RSD 3.8%) and repeatability (RSD 2%) and its high extraction frequency (up to 5 h(-1)) makes this method an inexpensive, high precision and fast tool for monitoring (99)Tc in environmental samples.

The second five years of sequential injection chromatography: significant developments in the technology and methodologies.

Sequential injection chromatography was proposed in 2003 to perform a simple, rapid, reagent-saving, environmentally benign, on-site, and instrumentally inexpensive separation procedure. Sequential injection chromatography is a version of sequential injection analysis, which is the second generation in the family of flow injection techniques. Despite its advantages over high-performance liquid chromatography, sequential injection chromatography has confronted some challenges. Furthermore, the applications of sequential injection chromatography in its first five years are almost all limited to pharmaceutical analysis. Interestingly, in its second five years, various developments in sequential injection chromatography technology were achieved. The developments have enhanced the efficiency of sequential injection chromatography and hence its applications have extended to biological, food, and environmental analyses. The main objectives of this review are to examine recent developments (2008-2013) in sequential injection chromatography and to describe how these developments improve the efficiency of the technology. The sequential injection chromatography methodologies reported during that period are also discussed along with controlling conditions and analytical results. The review also describes the principles, instrumentation, and procedure behind sequential injection chromatography.

Improved high-speed capillary electrophoresis system using a short capillary and picoliter-scale translational spontaneous injection.

Here, we describe an improved high-speed CE (HSCE) system using a short capillary and translational spontaneous sample injection. Several important factors for consideration in system design as well as various factors influencing the performance of the HSCE system were investigated in detail. The performance of this HSCE system was demonstrated in electrophoretic separation of FITC-labeled amino acids. Under optimized conditions, baseline separation of eight amino acids and FITC were achieved in 21 s with the plate heights ranging from 0.20 to 0.31 µm, corresponding to a separation rate up to 20 700 theoretical plates per second. The separation speed and efficiency of the optimized high-speed CE system are comparable to or even better than those reported in microchip-based CE systems.

Modified carbon paste sensor for the potentiometric determination of neostigmine bromide in pharmaceutical formulations, human plasma and urine.

A novel, simple, rapid, selective and sensitive method for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine) using four modified carbon paste electrodes was developed. Sensor 1 is based on ion-association Ns-TPB, sensor 2 used Ns-PT, sensor 3 comprises a mixture of (Ns-PT+Ns-TPB) and sensor 4 was constructed using (Ns-PT+ß-CD). Solvent mediator 2-NPPE exhibited a proper behavior including Nernstian slope ranging from 61.5±0.5 to 64.5±0.5 mV per decade over the pH range of 3.8-10 for the four sensors. Linear responses of Ns within the concentration range 1.0×10(-7)-1.0×10(-2) mol/L were obtained. The response time is very short (≤10s) with a detection limit 6.3×10(-8) M. In flow injection analysis (FIA), sensor 3 shows a Nernstian slope value 75.5±0.5 mV per decade within the concentration range of 1×10(-6)-1×10(-2) mol/L and with a detection limit 7.5×10(-7) mol/L. The utility of mixed or additives of ß-CD had a significant influence on increasing the sensitivity of sensors 3 and 4 compared to sensors 1 and 2. The sensors were applied for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine). The results obtained were satisfactory with excellent percentage recovery comparable with official method for the assay based on non-aqueous titration using perchloric acid as a titrant.

Compound coverage enhancement of electrospray ionization mass spectrometry through the addition of a homemade needle.

The response of many previously low-detectable or undetectable compounds in electrospray ionization mass spectrometry (ESI-MS) has been enhanced by the addition of a simple, homemade needle into the traditional ESI interface. The needle located between the ESI emitter and the ion sweep cone (inlet of the detector) would ionize those neutral gaseous compounds, formed during electrospray, by a corona discharge process. The mobile phases, ESI parameters and positions of the needle were investigated and optimized. Several groups of compounds and herbal extracts were tested using the homemade set-up. Both the results of the flow injection and the hyphenated MS analyses showed significant enhancement effects of our homemade needle. The advantages of the proposed method include low cost, simplicity and practicality.

Chip-based amperometric enzyme sensor system for monitoring of bioprocesses by flow-injection analysis.

A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 µAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.

Flow injection simultaneous determination of synthetic colorants in food using multiple pulse amperometric detection with a boron-doped diamond electrode.

A single-line flow injection system and multiple pulse amperometric detection using a boron-doped diamond electrode were employed to develop and optimize a simple, low-cost, and rapid method for the simultaneous determination of two pairs of food colorants: tartrazine and sunset yellow (TT-SY) or brilliant blue and SY (BB-SY). A dual-potential waveform was used: E(det.1)=-150 mV (400 ms duration) and E(det.2)=-450 mV (100 ms duration) vs. Ag/AgCl (3.0 mol L(-1) KCl). Polarization at E(det.1) or E(det.2) causes reduction of SY or the respective pair of colorants, TT-SY or BB-SY; hence, with proper current correction, both colorants in each pair can be determined. The obtained linear response ranges (detection limits) were 5.0-60.0 (2.5) and 1.0-50.0 (0.80) µmol L(-1), for TT and SY, or 5.0-60.0 (3.5) and 1.0-50.0 (0.85) µmol L(-1), for BB and SY, respectively. Investigation of possible interferents (other food colorants or additives) showed no significant interference with the methods here proposed, which were then used to simultaneously determine the pairs of colorants in industrialized food samples, with results that showed good agreement with those obtained using a comparative HPLC method.

Low-cost optical detectors and flow systems for protein determination.

Two miniature, fibreless, compact and highly integrated flow-through optoelectronic detectors dedicated for photometric and fluorimetric determination of proteins have been developed. Both detectors operating according to paired-emitter-detector-diode methodology are constructed only of light emitting diodes and therefore their total cost is extremely low. The photometric detector is dedicated for protein determination according to Bradford method based on detection of protein adduct with Coomassie Brilliant Blue. The fluorimetric detector allows determination of proteins after reaction with fluorescamine. Both developed detectors have been incorporated into economic flow systems constructed of microsolenoid valves and pumps. The resulting multicommutated/multipumping flow analysis systems enable detection of albumins and globulins at ppm levels, thus they are useful for protein determination in diluted samples of physiological fluids.

Indirect method for spectrophotometric determination of ascorbic acid in pharmaceutical preparations with 2,4,6-tripyridyl-s-triazine by flow-injection analysis.

A flow-injection indirect spectrophotometric method for the determination of ascorbic acid (AA) in pharmaceutical preparations is proposed. The method is based on the reduction of iron(III) to iron(II) by the AA, and by the subsequent reaction of the produced iron(II) with 2,4,6-tripyridyl-s-triazine (TPTZ) in buffered medium (pH=3.6) to form a coloured complex (λ(max)=593nm). The three-line manifold with one reaction coil was used. The linear range of the method is from 0.08 to 10µM of ascorbic acid, with the detection limit 24nM of AA. The proposed method is simple, rapid (sampling rate of 180 samples per hour), sensitive and reproducible (RSD 0.8%, n=100). The proposed method is very selective, because only the reducing substances with standard (formal) potentials lower than 0.6V would have the thermodynamic predisposition to interfere in the proposed method. Tested reducing substances (thiol compounds) did not give serious errors when present at the same concentrations as the ascorbic acid. The proposed method can be applied for the determination of AA in pharmaceutical preparations, down to picomolar quantity.