Fast and sensitive flow-injection mass spectrometry metabolomics by analyzing sample-specific ion distributions.

Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.

A protocol for studying the interaction between small-molecular drug and DNA using microdialysis sampling integrated with chemiluminescent detection.

It is of great significance to understand how drug molecules interact with DNA, which is one of the most important aspects of biological investigations in drug discovery at molecular level. Herein, with the model of ractopamine and calf thymus DNA (ct-DNA), a protocol using microdialysis (MD) sampling integrated with flow injection (FI)-chemiluminescent (CL) detection was developed for studying the interaction between small-molecular drug and DNA. After incubating ractopamine with ct-DNA, unbound ractopamine was on-line sampled using a MD probe, followed by being introduced into a FI-CL system for quantitation. The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe, and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters. The MD probe exhibited a mean recovery of 27.3% for ractopamine sampling under the optimal conditions. The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8 × 106 M-1 and 3.9 × 106 M-1, respectively, showing negligible difference. Ractopamine was estimated to have only one type of binding site on ct-DNA. The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.

Total polyphenols content in white wines on a microfluidic flow injection analyzer with embedded optical fibers.

Absorbance detection in food microdevices has not been thoroughly used due to low levels of sensitivity in measurements. Thus, it is necessary to develop microfluidic methods for improving photometric detection. For this purpose, a simple coupled-optical-fiber-polydimethylsiloxane (PDMS) microdevice was developed, to quantify polyphenols content in white wine employing the Folin-Ciocalteu reaction method. A 6V and 10W halogen lamp with an optical path length of 7mm between optical fibers, which were placed into the microchip, using guides at the outlet of the flow, increased the level of sensitivity during detection. The linear range was from 0.03mmol/L to 0.18mmol/L. Thus, the corresponding equation was: Abs=4.00(±0.16) [tannic acid]+0.17(±0.017). Intra-laboratory repeatability and reproducibility percentages were 2.95% and 6.84%, respectively. Such results were compared to those obtained from applying the conventional flow-injection analysis method, based on the same type of reaction. The relative error between methods was less than 13%.

On-line monitoring of blend uniformity in continuous drug product manufacturing process--The impact of powder flow rate and the choice of spectrometer: Dispersive vs. FT.

One of the commonly acknowledged issues in continuous manufacturing of drug products is how to provide a representative sampling on flowing powder to assure its blend uniformity. An investigation was conducted to improve understanding on the impact of powder flow rate under different continuous manufacturing conditions and the impact of optical parameters (such as resolution, co-adds, and integration time) on NIR spectral quality with respect to a dispersive and a Fourier transform instrument. A partial least squares (PLS)-based spectral pretreatment was found useful to tackle the impact of different flow rates on NIR spectral signals. Multivariate figures of merit (FOM) were used to evaluate performances across different instruments and optical settings and discover the advantageous selectivity and sensitivity on the Fourier transform than the dispersive instrument regardless of the use of co-adds.

Application of hot platinum microelectrodes for determination of flavonoids in flow injection analysis and capillary electrophoresis.

The determination of quercetin and rutin by flow injection analysis (FIA) and capillary electrophoresis (CE) using electrochemical detection was described. These flavonoids were determined at normal (unheated) and hot platinum microelectrodes using cyclic voltammetry. When quercetin or rutin is reaching the platinum electrode, a change of the current in the region of the platinum oxide formation is observed. Integration of the current changes in this in this region creates analytical signals in the form of peaks. An increase of temperature to about 76°C in a small zone adjacent to the microelectrode causes an increase of the analytical signal by more than 6 times under FIA conditions. This method enables the use of hot microelectrodes as detectors in HPLC or CE. In CE the improvement of the analytical signal at hot microelectrodes is smaller than in FIA and increase only 1.3-3.4 times. Heated microelectrodes were used for analysis of the flavonoids in natural samples of the plant (extract of sea buckthorn) and a pharmaceutical preparation (Cerutin).

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis.

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 µL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9h(-1), a linear calibration range of 0.5-15 mg L(-1) (absorbance=5.40×10(-2) [acetaldehyde, mg L(-1)], R(2)=0.998), a relative standard deviation (RSD) of 1.90% (n=10, acetaldehyde concentration of 2.5 mg L(-1)), and a limit of detection (LOD) of 12.3 µg L(-1). The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD=93.0 µg L(-1) and sampling rate=4 h(-1)). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6-101.9%), comparable to those obtained by GC (96.4-102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.

High-resolution quantitative metabolome analysis of urine by automated flow injection NMR.

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) (1)H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 µL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ~20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Suppression of peptide sample losses in autosampler vials.

Protein or peptide sample losses could accompany all steps of the proteomic analysis workflow. We focused on suppression of sample adsorptive losses during sample storage in autosampler vials. We examined suppression capabilities of six different sample injection solutions and seven types of autosampler vial surfaces using a model sample (tryptic digest of six proteins, 1 fmol per protein). While the vial material did not play an essential role, the choice of appropriate composition of sample injection solution reduced adsorptive losses substantially. The combination of a polypropylene vial and solution of poly(ethylene glycol) (PEG) (0.001%) or a mixture of high concentrated urea and thiourea (6 M and 1 M) as injection solutions (both acidified with formic acid (FA) (0.1%)) provided the best results in terms of number of significantly identified peptides (p < 0.05). These conclusions were confirmed by analyses of a real sample with intermediate complexity (in-gel digest from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Addition of PEG into the real sample solution proved to prevent higher losses, concerning mainly hydrophobic peptides, during up to 48 h storage in the autosampler in comparison with a formic acid solution and even with a solution of highly concentrated urea and thiourea. Using PEG for several months was not accompanied by any adverse effect to the liquid chromatography system.

Evaluation of carbon nanotubes as chiral selectors for continuous-flow enantiomeric separation of carvedilol with fluorescent detection.

Single-walled carbon nanotubes (SWNT) are proposed as chiral selectors for separation of carvedilol stereoisomers beginning since its racemic mixture. The novel developed FIA-methodology employs a microcolumn (mC) packed with a few milligrams of SWNT which showed to be effective in S(-) and R(+) carvedilol separation. Attending to spectral properties of analytes, molecular fluorescence was employed in the detection step. Separation of carvedilol enantiomers was achieved in less than 70s with an acceptable resolution factor of 3.16. Variables that influence the chiral separation such as pH and composition of eluent solution, sample injection volume and flow rate, activation mode of NTs and mass of the same in column have been examined in detail. At optimal operational conditions, well repeatability was achieved using the same column for more than 100 injections, putting in evidence the stability of nanomaterial and the efficacy and versatility of the proposed FIA-configuration. The new methodology was successfully applied to S(-) and R(+) carvedilol quantification in pharmaceutical preparations, resulting an attractive alternative to traditional separative methods being fast, simple, using low cost instrumentation and producing scarce waste.

Serum alkaline phosphatase assay with paired emitter detector diode.

A simple multicommutated flow system based on optoelectronic detector, three valves and peristaltic pump only has been developed for photometric determination of alkaline phosphatase activity in human serum. A miniaturized, compact flow-through detector dedicated to selective photometric detection of product formed in the course of the enzyme assay has been constructed using two paired light emitting diodes. The proposed analytical procedure based on kinetic methodology of enzyme activity detection and stopped-flow methodology of two-point measurements eliminates interferences caused by intense color of real samples and impurities present in commercial reagents. After optimization the system allows reproducible, mechanized analysis of human serum in relatively short time (8-9 samples per hour). Volume of serum required for single determination is 0.05mL only. The system validated with real clinical samples is useful for determination of enzyme activity in human serum at physiological and pathological levels.

Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations.

Application of a sensitive and rapid flow injection analysis (FIA) method for determination of topiramate, piracetam, and levetiracetam in pharmaceutical formulations has been investigated. The method is based on the reaction with ortho-phtalaldehyde and 2-mercaptoethanol in a basic buffer and measurement of absorbance at 295 nm under flow conditions. Variables affecting the determination such as sample injection volume, pH, ionic strength, reagent concentrations, flow rate of reagent and other FIA parameters were optimized to produce the most sensitive and reproducible results using a quarter-fraction factorial design, for five factors at two levels. Also, the method has been optimized and fully validated in terms of linearity and range, limit of detection and quantitation, precision, selectivity and accuracy. The method was successfully applied to the analysis of pharmaceutical preparations.

A luminescence study of the interaction of sulfobutylether-ß-cyclodextrin with rutin.

The luminol/sulfobutylether-ß-cyclodextrin (SBE(7M)-ß-CD) chemiluminescence (CL) system and the interaction of SBE(7M)-ß-CD/rutin were first described by flow injection (FI) CL method. It was found that SBE(7M)-ß-CD with luminol could form 1:1 complex online, which could accelerate the electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL intensity of luminol. The enhancement of CL intensity was proportional to the concentrations of SBE(7M)- ß-CD with a linear range from 25 to 1750 µmol L⁻¹. It was also found that rutin could inhibit the CL intensity from luminol/SBE(7M)-ß-CD system, and the decrement of CL intensity was logarithm over the concentrations of rutin ranging from 0.1 to 100.0 nmol L⁻¹, giving the regression equation ΔI = 32.90lgC(rutin) + 16.26 (R² = 0.9952) with a detection limit of 0.03 nmol L⁻¹ (3σ). According to the proposed CL model, the binding constant (K(CD-R)) and the stoichiometric ratio of SBE(7M)-ß-CD/rutin complex were obtained as 1.6 × 106 L² mol⁻² and 2:1. The possible mechanism of luminol/SBE(7M)-ß- CD/rutin interaction was also discussed. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE(7M)-ß-CD/rutin complex, with a total excretion of 68.8% within 8.0 h.

Automatic determination of chlorine without standard solutions using a biamperometric flow-batch analysis system.

This study presents an automatic analysis system that does not require the use of standard solutions. The system uses an electrochemical flow cell for in line generation of the standards, and operates under the standard addition technique. The versatility of this system was demonstrated by the development of a one key touch fully automatic method for the determination of total available chlorine in real samples. The extremely simple, accurate and inexpensive method was based simply on the biamperometric monitoring of the well known redox reaction of chlorine with iodide ions in a flow-batch system, where the produced iodine (triiodide ions) generates an electrical current proportional to the chlorine concentration in the sample. The flow-batch parameters were optimized to maximize the sensitivity without losses on the precision of the analysis. An excellent linear dependence between the biamperometric signal and the chlorine concentration for the standard additions and a good agreement between the proposed approach and a reference method were obtained. The method was successfully applied to determine chlorine in several different bleach and chlorinated water samples (r=0.9995, LOD=8.261 x 10(-7) mol L(-1)) and could be easily extended to other oxidants and samples. Comparison to a reference method and recoveries close to 100% demonstrated the reliability of the proposed method. In addition, low residue disposal and reagent consumption, allied with high accuracy and precision, make it very promising for routine applications.

Determination of arsenic in foods by flow injection on-line sorption pre-concentration with hydride generation atomic fluorescence spectrometry.

A method was developed for the determination of total arsenic in foods using flow injection on-line sorption coupled with hydride generation atomic fluorescence spectrometry (HG-AFS) using a cigarette filter as the sorbent material. After reducing As(V) to As(III) by using L-cysteine, the determination of total arsenic was achieved through on-line formation and retention of the pyrrolidine dithiocarbamate arsenic complex (As(III)-PDC) on the cigarette filter, which was packed in the pre-concentration column and total arsenic was determined by HG-AFS. The analytes were eluted with 1.68 mol l(-1) HCl from the sorbent material. With consumption of 22 ml of the sample solution, an enrichment factor of 25.6 was obtained at a sample throughput of 11.6 h(-1). The detection limits (3 standard deviations) and the precision (relative standard deviation) in foods ranged from 2.5 to 9.9 ng g(-1) and from 1.1 to 2.2%, respectively. The method was used to determine arsenic in carrot, mushroom, chicken tissue, cod fish, rice, common carp and shrimp.

A single standard calibration module for flow analysis systems based on solenoid microdevices.

Only two computer-controlled microsolenoid devices, namely two micropumps or one micropump and one microvalve, are sufficient for the construction of on-line dilution modules useful in several flow analytical systems for the calibration using single standard. Three simple constructions of such modules were tested and compared. The most promising is the one based on the concept of a microvalve controlling dilution ratio of the standard and a solenoid micropump playing a double role: solution pumping device and mixing segments homogenizer. All investigated modules were tested with paired emitter detector diode (PEDD) as photometric flow-through detector and bromothymol blue as a model analyte. The best module was implemented into more advanced flow-injection system dedicated for optical detection of alkaline phosphatase activity using UV-PEDD-based flow-through detector for the enzyme reaction product.

Principle, calibration, and application of the in situ alkali chloride monitor.

The extended use of biomass for heat and power production has caused increased operational problems with fouling and high-temperature corrosion in boilers. These problems are mainly related to the presence of alkali chlorides (KCl and NaCl) at high concentrations in the flue gas. The in situ alkali chloride monitor (IACM) was developed by Vattenfall Research and Development AB for measuring the alkali chloride concentration in hot flue gases (less than or approximately 650 degrees C). The measurement technique is based on molecular differential absorption spectroscopy in the UV range. Simultaneous measurement of SO(2) concentration is also possible. The measuring range is 1-50 ppm for the sum of KCl and NaCl concentrations and 4-750 ppm for SO(2). This paper describes the principle of the IACM as well as its calibration. Furthermore, an example of its application in an industrial boiler is given.

Critical evaluation of novel dynamic flow-through methods for automatic sequential BCR extraction of trace metals in fly ash.

Two novel dynamic extraction approaches, the so-called sequential injection microcolumn extraction and sequential injection stirred-flow chamber extraction, based on the implementation of a sample-containing container as an external extraction reactor in a sequential injection network, are for the first time, optimized and critically appraised for fractionation assays. The three steps of the original Community Bureau of Reference (BCR) sequential extraction scheme have been performed in both automated dynamic fractionation systems to evaluate the extractability of Cr, Cu, Ni, Pb, and Zn in a standard reference material of coal fly ash (NIST 1633b). In order to find the experimental conditions with the greatest influence on metal leachability in dynamic BCR fractionation, a full-factorial design was applied, in which the solid sample weight (100-500 mg) and the extraction flow rate (3.0-6.0 mL min(-1)) were selected as experimental factors. Identical cumulative extractabilities were found in both sequential injection (SI)-based methods for most of assayed trace elements regardless of the extraction conditions selected, revealing that both dynamic fractionation systems, as opposed to conventional steady-state BCR extraction, are not operationally defined within the selected range of experimental conditions. Besides, the proposed automated SI assemblies offer a significant saving of operational time with respect to classical BCR test, that is, 3.3 h versus 48 h, for complete fractionation with minimum analyst involvement.

Determination of preservatives in meat products by flow injection analysis (FIA).

Various preservatives are added to meat products to extend shelf-life and enhance food safety; thus, their determination is essential for legislative purposes and consumer health. Analytical methodologies based on flow injection analysis (FIA) offer attractive advantages compared with other procedures, such as versatility, precision, low cost, speed and ease of automation. This review considers the status of published FIA methodologies for the determination of preservatives in meat products. The techniques are described regarding their application to different preservatives (nitrates and nitrites, sulfites, sorbates, benzoates and p-hydroxybenzoate esters), with emphasis on extraction, separation, detection and quantification procedures in meat matrices.

Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system.

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB-GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (lambda=412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 microM, with detection limits of 0.031 and 0.014 microM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.)<5.0% (n=10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.

Spectrophotometric determination of chloride in waters using a multisyringe flow injection system.

A multisyringe flow injection system (MSFIA) with spectrophotometric detection is proposed as a fast, robust and low-reagent consumption system for the determination of chloride (Cl(-)) in waters. The system is based in the classic reaction of Cl(-) with Fe(3+) and Hg(SCN)(2), but due to the hazardous properties of this last reagent, the proposed methodology has been developed with the aim to minimize the consumption of this one, consuming less than 0.05 mg of Hg for a Cl(-) determination, being the system of this type with the lowest Hg consumption. The linear working range was between 1 and 40 mg L(-1) Cl(-) and the detection limit was 0.2 mg L(-1) Cl(-). The repeatability (RSD) was 0.8% for a 10 mg L(-1) Cl(-) solution, and the injection throughput was 130 h(-1). The proposed system is compared with other chloride monitoring flow systems, this comparison is realized with a point of view of the equilibrium between the obtained analytical features and produced residues toxicity. The proposed system was applied to the determination of Cl(-) in mineral, tap and well water.