Stopped-flow Mode Research Articles

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC-ESI-MS/MS and stopped-flow HPLC-NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid-liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C(18) reversed-phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI-MS/MS detection in an on-line mode or (1)H-NMR (500 MHz) spectroscopic detection in a stopped-flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12-hydroxy-ilaprazole sulfide (M2), 11,12-dihydroxy-ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC-ESI-MS/MS and HPLC-NMR could be widely applied in detection and identification of novel metabolites.

Sensor array and stop-flow mode applied to discrimination and quantification of gas mixtures

The presented work focused on the strategy which offers widening the scope of applicability of sensor systems. The strategy was based on: (1) employing sensor array, which consists of many sensors, (2) applying stop-flow as the mode of sensor array operation, (3) utilizing response values of selected sensors measured at the same time point, (4) using only small fraction of obtained results. The performance of the system was evaluated with reference to the problems of discrimination of gas mixtures and quantification of their components. A considerable experimental material was the basis for our study. It comprised measurement results of 250 two-component VOC mixtures in air. They represented 10 classes characterized by fixed qualitative composition and variable quantitative one. The results of our work proved that sensor system based on big sensor array and operated in stop-flow mode has wider scope of application than the system utilizing small sensor array and one kind of exposure conditions.

The stop-flow mode of operation applied to a single chemiresistor

The stop-flow mode of operation was characterized as applied to a chemiresistor. In this mode, the main factor causing the alteration of exposure and operation conditions was the flow of gas. A chemiresistor was represented by the pool of 15 different TGS sensors. They were exposed to samples of air containing an analyte together with an interfering compound. The characteristics of the mode consisted of the following elements: (1) the analysis of sensor output signal, (2) the analysis of time variability of classification performance regarding the interfering compounds (3) the analysis of time variability of quantification error regarding the analyte. In order to characterize stop-flow mode the single-sensor perspective was adopted, i.e., classification and quantification models utilized time-point responses of single sensors. It was shown that exposure and operation conditions were well reflected in chemiresistor response, which showed a specific time-pattern. Interestingly, the distribution of classification useful information in sensor response was not related to sensor operation conditions in a fixed manner. It differed between sensors and gases. Contrarily, the distribution of quantification-useful information in sensor response was found specific for the stop-flow mode, irrespective of the sensor and the analyte. It was concluded that the operating conditions may be used as a means of shaping the content of quantification and classification useful information in sensors responses. The obtained results may serve as a guideline when considering the application of stop-flow as the mode of sensor operation.

Application of LC–NMR to the identification of bulk drug impurities in NK1 antagonist GW597599 (vestipitant)

Liquid chromatography-NMR (LC-NMR) spectroscopy was used to obtain detailed information regarding the structure of the major bulk drug impurities present in GW597599 (vestipitant). The one-dimensional (1)H LC-NMR experiments were performed in both continuous and stop-flow modes on a sample of GW597599 (vestipitant) enriched with mother liquor impurities. The information derived from both LC-NMR and LC-MS data provided the structural information of all major impurities. The full characterisation of the impurities by high-resolution NMR spectroscopy was ultimately performed on appropriately synthesised compounds.

Dynamic Peptide Folding and Assembly for DNA Separations

The phenomenon of counterion-mediated DNA-condensation is fundamental to most DNA related activity in the cell, from chromosome packaging to control over translational mechanisms. Developing synthetic systems to manipulate DNA-condensation is essential for the development of biotechnologies for gene encapsulation and DNA-separation. We investigate the dynamics of the DNA-condensation by using our model peptide where the interaction between DNA and peptide is non-specific. We have designed a peptide that shows switchable surface activity, where the folded form of the peptide is amphiphilic and the unfolded form is not amphiphilic. The peptide is α-helical, containing 23 amino acids with variation in the number and distribution of hydrophobic and charged amino acids. The designs incorporate hydrophobic residues on one side (leucines and alanines) and hydrophilic residues on the opposite side so that helix is surface active. The secondary structure has been characterized by using circular dichroism spectropolarimetry, and we show that the peptide has a transient secondary structure as a function of monovalent salt concentration. The behavior of the peptide at air-water interface is characterized by pendant drop/bubble method and modeled accordingly. Our hypothesis is that the unfolded peptide is in equilibrium with the folded peptide in the bulk solution but in presence of DNA, the unfolded peptide folds and then binds to DNA. Critical Aggregate concentration of the peptide for DNA condensation is determined by using multi angle light scattering, which is also used to calculate the radius of gyration and molecular weight of these condensates. We investigate the kinetics of the condensation process by using Circular dichroism in Stop-flow mode and also by isothermal titration calorimetry.

LC-NMR identification of a novel taurine-related metabolite observed in 1H NMR-based metabonomics of genetically hypertensive rats

This paper describes the LC-NMR spectroscopic identification of a novel urinary endogenous metabolite responsible for the signals, which were found as major contributors to the separation between genetically hypertensive rats (SHRSP) and normotensive control rats (WKY) in previous NMR-based metabonomic studies. Urine samples from 26-week-old normotensive rats were analyzed by an LC-NMR system equipped with a reversed-phase column having high retention ability for polar compounds. 1H NMR spectra were continuously obtained in the on-flow mode, and the retention times of the unassigned signals in question were determined. Various two-dimensional spectra were subsequently measured for the fraction containing the unassigned signals under the stop-flow mode, which enables for a long accumulation resulting in the enhancement of signal-to-noise ratios. The candidate compound obtained from these LC-NMR data was synthesized, and the NMR and mass spectra were compared with those of the LC-NMR fraction. The unknown metabolite was identified as succinyltaurine from these experiments together with standard addition experiments. This novel metabolite, which is characteristic of the normotensive rats, is very interesting because it is structurally related to hypotensive taurine, and not substantially detected in the genetically hypertensive rats, which excreted more taurine than the normotensive rats. The biological and pathophysiological significances of succinyltaurine remain to be investigated.

Anti-fungal effect of berberine on Candida albicans by microcalorimetry with correspondence analysis

Using a LKB-2277 bioactivity monitor, stop-flow mode, the power–time curves of Candida albicans growth at 37 °C affected by berberine were measured. The check experiments were studied based on agar cup method to observe the inhibitory diameter and serial dilution method to determine the minimal inhibitory concentration (MIC) of berberine on C. albicans growth. By analyzing the quantitative thermogenic parameters taken from the power–time curves using correspondence analysis (CA), we could find that berberine at a low concentration (5.0 μg mL−1) began to inhibit the growth of C. albicans and at a high concentration (75.0 μg mL−1) completely inhibited C. albicans growth. The anti-fungal activity of berberine could also be expressed as half-inhibitory concentration IC50, i.e., 50% effective in this inhibition. The value of IC50 of berberine on C. albicans was 34.52 μg mL−1. The inhibitory diameters all exceeded 10 mm in test range and the MIC was 500 μg mL−1. Berberine had strong anti-fungal effect on C. albicans growth. This work provided an important idea of the combination of microcalorimetry and CA for the study on anti-fungal effect of berberine and other compounds. Compared with the agar cup method and serial dilution method, microcalorimetry not only offered a useful way for evaluating the bioactivity of drugs, but also provides more information about the microbial growth and all this information was significant for the synthesis and searching of antibiotics.

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis-Menten constant (K(M)) of the enzyme and Vmax of the reaction. The obtained K(M) of the reaction was 14 +/- 3 g L(-1) (41 microM). Furthermore, inhibition by adenosine 5'-monophosphate (AMP) was evaluated, and the K(M)(App) value was determined to be 12 +/- 2 g L(-1) (35 microM) for 7.5 and 15 microM AMP, respectively, with Vmax decreasing from 0.1 +/- 0.03 to 0.05 +/- 0.01 g L(-1) min(-1). Therefore, AMP exerted a non-competitive inhibition.

On-Chip Immunoassay Using Electrostatic Assembly of Streptavidin-Coated Bead Micropatterns

We propose an original concept for a sandwich immunoassay that is completely performed on-chip using streptavidin-coated beads as substrate. The latter are electrostatically self-assembled on aminosilane micropatterns at the bottom of a microfluidic channel. We use mouse IgG diluted in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) solution as target antigen. The fluorescent sandwich immunocomplex is formed on the beads during the operation of the chip both in stop-flow and continuous-flow modes. Target mouse IgG antigen is detected down to a concentration of 15 ng/mL in stop-flow mode and 250 pg/mL in continuous-flow mode, using only 1300 nL of sample volume. We also demonstrate the possibility of simultaneous detection of two different antigens in a PBS-BSA solution using a dual microfluidic channel structure.

Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis

This study investigated the anti-fungal activity of coptisine on Candida albicans growth. The metabolic power-time curves of Candida albicans growth at 37 degrees C affected by coptisine were measured by microcalorimetry using an LKB-2277 Bioactivity Monitor with stop-flow mode. Then, the diameter of inhibitory zones in the agar layer was observed using agar cup method, and the minimal inhibitory concentration (MIC) of coptisine on Candida albicans growth was determined by serial dilution method. From the principal component analysis on nine quantitative parameters obtained from the power-time curves, we could easily evaluate the anti-fungal activity of coptisine by analysing the change of values of the main two parameters, growth rate constant k and maximum power output in the log phase P(m, log). The results showed that coptisine had strong anti-fungal activity: at a low concentration (45 microg ml(-1)) began to inhibit the growth of Candida albicans and at a high concentration (500 microg ml(-1)) completely inhibited Candida albicans growth. Coptisine gave big inhibitory zones with diameters between 11 and 43 mm within test range, and the MIC of it was 1000 microg ml(-1). Coptisine had strong anti-fungal activity on Candida albicans growth. The method of microcalorimetry applied for the assay of anti-fungal activity of coptisine was quantitative, sensitive and simple. This work will provide useful information for the development of chemical biology policy in the use of anti-microbials in food and drug production.

Sandwich immunoassay on a microfluidic chip using patterns of electrostatically self-assembled streptavidin-coated beads

In this paper, we demonstrate a bead-based microfluidic immunoassay using self-assembled streptavidin-coated bead micropatterns. The beads are assembled on the microfluidic channel surface using electrostatic forces between the negatively-charged streptavidin-coated beads and the positively-charged aminosilane micropatterns. The immunoassay is performed in the stop-flow mode and uses mouse IgG diluted in phosphate buffered saline (PBS) solution as the model target antigen. The total immunoassay takes around 30 min and is able to detect mouse IgG antigen down to a concentration of 15 ng/mL by using only 560 nl of analyte volume.

Stop‐flow comprehensive two‐dimensional gas chromatography with pneumatic switching

A new method for performing comprehensive GC x GC in the stop-flow mode is presented. A device was used to pneumatically stop the flow in the first dimension ((1)D) (by applying pressure pulses at the junction between the two columns), while flow was maintained in the second dimension ((2)D). This allowed for better preservation of resolution in the (1)D of the GC x GC chromatograms, and the extension of the (2)D's separation space, reducing or eliminating the extent of wraparound. When increased flow rates in the (2)D were used, sensitivity enhancements were also observed.

Flow injection determination of total chromium in urine of occupationally exposed workers

An efficient and fast method has been developed to determine chromium in urine. It is based on an on-line ultrasound-assisted sample digestion procedure exploiting the stopped-flow mode, followed by flow injection chromium preconcentration using a minicolumn filled with a commercially available chelating resin (Chelite Che). The precision as repeatability (expressed as relative standard deviation, n = 11) was 1.4% at a 10 µg L−1 level. The recovery, based on analysis of spiked samples and solutions including urine matrix components, is quantitative. The detection limit was 0.5 µg L−1 when flame atomic absorption spectrometry was employed. The preconcentration factor is 42.6. The method was applied to the determination of chromium in urine of workers exposed to welding fumes.

Determination of trace metals in urine with an on-line ultrasound-assisted digestion system combined with a flow-injection preconcentration manifold coupled to flame atomic absorption spectrometry

A flow analysis method with on-line sample digestion/minicolumn preconcentration/flame atomic absorption spectrometry is described for the determination of trace metals in urine. First, urine sample was on-line ultrasound-assisted digested exploiting the stopped-flow mode, and then the metals were preconcentrated passing the pre-treated sample through a minicolumn containing a chelating resin. A home-made minicolumn of commercially available imminodiacetic functional group resin, Chelite Che was used to preconcentrate trace metals (Cu, Fe, Mn and Ni) from urine. The proposed procedure allowed the determination of the metals with detection limits of 0.5, 1.1, 0.8 and 0.8 μg L −1, for Cu, Fe, Mn and Ni, respectively. The precision based on replicate analysis was less than ±10.0%, and the enrichment factor obtained was between 21.3 (Mn) and 44.1 (Ni), for sample volumes between 2.5 and 5.0 mL, and an eluent volume of 110 μL. This procedure was applied for determination of metals in urine of workers exposed to welding fumes and urine of unexposed persons (urine control).

Acoustic resonances in straight micro channels: Beyond the 1D-approximation

Acoustic actuation can be used to perform several tasks in microfluidic systems. In this paper, we investigate an acoustic separator through micro-PIV analysis in stop-flow mode and numerical simulations, and a good agreement between the two is found. Moreover, we demonstrate that it is not sufficient only to characterize devices in flow-through mode, since in these systems much different resonant patterns can result in similarly looking band formations. Furthermore, we conclude that extended 1D approximations of the acoustic radiation force are inadvisable, and instead, a 2D model is preferred. The results presented here provide valuable insight into the nature and functionality of acoustic microdevices, and should be useful in the interpretation and understanding of the same.

Rapid determination of hydroflumethiazide in dosage forms by time-resolved chemiluminescence

Hydroflumethiazide was determined from the chemiluminescence produced in its reaction with Ce(IV) in acidic medium containing rhodamine 6G as a sensitizer using flow injection techniques (stopped flow mode). A straightforward automatic method based on measuring the peak height and peak area, which are directly proportional to the hydroflumethiazide concentration, was thus developed. The calibration graphs were linear over the concentration range of 1.0–30 µg mL−1. The limit of detection as determined according to Clayton was 0.97 and 0.91 µg mL−1 for peak height and peak area measurements, respectively. The relative standard deviation for ten samples was less than 3.0% with both types of measurements. The procedure was applied to the determination of hydroflumethiazide in pharmaceutical formulations, yielding excellent recoveries, since the determination is free of interference from common excipients and other drugs which are present in the formulation.

Simultaneous spectrophotometric determination of copper(II) and zinc(II) based on their kinetic separation in flow-injection systems

Flow-injection methods are developed for the spectrophotometric analysis of binary copper(II) and zinc(II) mixtures. They are based on measurements of a differential kinetic signal caused by ligand-exchange reactions that occur in the flow between the complexes of these metals with the same chromogenic reagent (4-(2-pyridylazo)-resorcinol or zincon) and aminopolycarboxylic acids. Two different approaches are used for the kinetic separation (or masking) of these metals followed by the on-line processing of the recorded signal by regression analysis. One of them is monitoring an indicator reaction in the stopped-flow mode, and the other is recording the separated peaks in the flow-injection system with two reaction zones reaching the detector over certain periods. The optimum detection conditions were found (cmin = 0.03 μg/mL), which allow the detection of the studied metal ions in mixtures in a ratio of no more than 1: 5 with a relative error of no more than 5%, good precision (RSD < 10%, n = 6, P = 0.95), and high throughput (90 h−1). The developed procedures were tested in the analysis of model mixtures and pharmaceutical preparations.

Monitoring of the smoking process by multicommutation Fourier Transform Infrared spectroscopy

Nicotine was selected as the target molecule for monitoring of the smoking process by multicommutation Fourier Transform Infrared spectroscopy (FTIR). The method involved the use of CHCl 3 for on-line extraction of nicotine from tobacco, cigarette filters and tobacco ash from NH 4OH alkalinized samples, and absorbance measurement of the characteristic band at 1316 cm −1 in the stopped-flow mode, by obtaining the peak area in the range between 1334 and 1300 cm −1. Under the best operational conditions, the procedure developed provided a detection limit of 0.05 mg mL −1 nicotine, corresponding to 0.5 mg g −1 in the solid sample, a relative standard deviation less than 2.5%, and a sampling frequency of 12 determinations h −1. It can be concluded that nicotine migrates in the smoke mainstream towards the filter during the smoking process. The smoking of cigarettes and cigars is different. Nicotine is retained weakly by both tobacco and filter in the case of cigarettes, and strongly by the unburned tobacco in cigars. The incomplete smoking of cigars and cigarettes reduces nicotine intake and thus reduces the additive effect.

Single and Multiphase Catalytic Oxidation of Benzyl Alcohol by Tetrapropylammonium Perruthenate in a Mobile Microreactor System

A mobile microreactor system, with flow and temperature control for organic synthesis, is described. The system can be used anywhere a venting outlet is available. The microreactors can be operated in different flow patterns (continuous flow, stop-flow, or programmed-flow) providing reaction times from a few minutes to a few hours. The system was tested for the catalytic oxidation of benzyl alcohol to benzaldehyde by tetrapropylammonium perruthenate (TPAP) with N-methyl-morpholine-N-oxide in the liquid phase under stop-flow mode and on supported TPAP with oxygen under continuous flow mode. The conversion of benzyl alcohol in the microreactor was close to that of a small batch reactor for the liquid phase reaction. For the multiphase reaction, a conversion of 30–40 % was obtained with residence times below 1 min.

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography × liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ( 2D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ( 1D) column can be derived. Theory clearly indicates that the choice of the 1D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.