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PubMed2003

Determination of organic peroxides by liquid chromatography with on-line post-column ultraviolet irradiation and peroxyoxalate chemiluminescence detection.

A HPLC method was developed for the simultaneous determination of organic peroxides and hydrogen peroxide with peroxyoxalate chemiluminescence (PO-CL) detection following on-line UV irradiation. Organic peroxides [i.e., benzoyl peroxide (BP), tert.-butyl hydroperoxide (BHP), tert.-butyl perbenzoate (BPB), cumene hydroperoxide (CHP)] were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide, which was determined by PO-CL detection. The conditions for UV irradiation and PO-CL detection were optimized by a flow injection analysis (FIA) system. Generation of hydrogen peroxide from peroxides with on-line UV irradiation also was confirmed by the FIA system by incorporating an enzyme column reactor immobilized with catalase. The separation of four organic peroxides and hydrogen peroxide by HPLC was accomplished isocratically on an ODS column within 30 min. The detection limits (signal-to-noise ratio=3) were 1.1 microM for hydrogen peroxide, 6.8 microM for BP, 31.3 microM for BHP, 7.5 microM for BPB and 1.3 microM for CHP. The proposed method was applied to the determination of BP in wheat flour.

Calibration↗
PubMed2003

Adduct formation in electrospray ionization mass spectrometry II. Benzoic acid derivatives.

This work serves as a follow-up to Part I of experiments designed to determine the underlying principles in the formation of pseudomolecular, or adduct, ions during electrospray ionization. Aromatic acids were studied by flow injection analysis in the negative ionization mode of electrospray ionization mass spectrometry. Part I dealt with common acidic anti-inflammatory pharmaceuticals. such as ibuprofen and related analogues. Part II deals with functionally less complex molecules, namely benzoic acid (BA) and substituted benzoic acids. Halide-substituted molecules are investigated to deduce the effect of electron-withdrawing substituents (bromo-, chloro-, and fluoro-) and ring position (ortho-, meta- and para-) on the response of a traditional deprotonated molecular ion ([M-H]-) and a sodium-bridged dimer ion ([2M-2 H+Na]-). Amino-substituted benzoic acids are also analyzed in order to study the effect of an additional ionizable group on the molecule, and para-tert.-butyl-BA is analyzed to study the effect of increased hydrophobicity, as they relate to the formation of pseudomolecular ions. This study shows that solution character [octanol-water partition coefficient (or log P) and pKa] of the model compounds controls the relative efficiency of formation of [M-H]- and [2M-2H+Na]- ions. However the relative gas phase character (gas phase basicity and proton affinity) also has a significant effect on the formation of the sodium-bridged dimer ion. For the halide-substituted species, placement of the electron-withdrawing atom at the meta-position gives the greatest enhancement in sensitivity. Observations also show that as the structural complexity of the model compound increases, predictions relating analyte acidity to sodium-bridged dimer ion formation give way to a stronger dependence between log P values and ionization efficiency. Supporting this hypothesis is the nearly ten-fold enhancement in signal for tert.-butyl BA relative to BA. due to the greater hydrophobicity, and consequently, increased surface activity in an electrosprayed droplet of the analyte molecule.

Benzoic Acid↗
PubMed1997

Determination of staphylococcal enterotoxin B by on-line (micro) liquid chromatography-electrospray mass spectrometry.

The use of (micro) liquid chromatography electrospray mass spectrometry (LC-ES-MS) was investigated as potential technique for the determination of the high-molecular-mass protein toxin Staphylococcal Enterotoxin B (SEB). The molecular mass was determined (28,366.3 +/- 1.1) by flow injection analysis and micro-LC-MS with a TSK-gel Phenyl-5PW column packing. Both methods allowed molecular-mass determination of SEB at levels down to 3 pmol/ml. Additional evidence of identification was obtained by detecting the presence of a disulfide bridge by the addition of 2-mercaptoethanol and by tryptic digestion. Collision induced dissociation spectra were recorded from the major tryptic fragments resulting in a sufficient number of sequence ions to allow for the determination of the amino acid sequence. On the analysis of the tryptic digests with C18 reversed-phase columns the use of micro-LC-MS-MS resulted in an 30-40-fold increase in sensitivity as compared with conventional-size LC-MS-MS.

Amino Acid Sequence↗
PubMed1998

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning.

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Anthracenes↗
PubMed1997

Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

Animals↗
PubMed1999

Efficient isolation of the rare diarrhoeic shellfish toxin, dinophysistoxin-2, from marine phytoplankton.

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Animals↗
PubMed2001

Characterization of raw water for the ozone application measuring ozone consumption rate.

This study was conducted to illustrate an ideal method for characterizing natural waters for ozonation processes in drinking water treatment plants. A specific instrument designed with the flow injection analysis (FIA) technique enabled us to measure accurately the ozone decomposition rate, which was found to consist of two stages: the instantaneous ozone consumption stage and the slower ozone decay stage. The ozone consumption rate was measured at the initial and secondary stages by determining certain parameters called the instantaneous ozone demand (ID) and the pseudo first-order decay rate constant (k(c)). Using the OH*-probe, the yield of OH* per consumed ozone was also measured to determine its potential to produce OH* for the oxidation of micropollutants during the ozonation process. The ozone consumption of the ID values was significant in most natural waters, and substantial amounts of OH* were found to generate during the instantaneous ozone consumption stage. This study also investigated the effects of particulates, ozone doses, and sequential ozone injection on ozone decomposition kinetics and OH* formation yield.

Algorithms↗
PubMed2002

Characterization of ozone decomposition in a soil slurry: kinetics and mechanism.

A series of soil slurry experiments were performed in a carefully conceived reactor set-up to investigate the characteristics of the catalytic decomposition of ozone on a sand and iron surface. Real time on-line monitoring of ozone in the reaction module was possible using flow injection analysis coupled with a computer-controlled UV detector and data acquisition system. The effects of the soil media and size, ozone dosage, pH and p-CBA as a probe compound were examined at the given experimental conditions. Two apparent phases existed, and ozone instantaneously decomposed within one second in the first phase. These were defined as the instantaneous ozone demand (ID) phase, and the relatively slow decay stage. The interactions of ozone with the soil organic matter (SOM) and metal oxides were attributed mostly to the instantaneous decomposition of ozone. From the probe (p-CBA) experiments, 60-68% of total p-CBA removal occurred during the ID phase. The generation of hydroxyl radicals (OH.) was demonstrated and was closely related with metal oxides as well as SOM. Metal oxides in soil surface were considered to have relatively faster reaction rate with ozone and provide more favorable reactive sites to generate higher amount of OH. than SOM. Even at one-tenth concentration of the sands, a goethite-induced catalytic reaction outfitted the removal rate ofp-CBA among all the soils tested. More than 40% of total p-CBA removal occurred on the soil surface. It was inferred that the radical reaction with the probe compound seemed to take place not only on the soil surface but also in the solid-liquid interface. Ozone decomposition and the reaction between OH. and p-CBA appeared to be independent of any change in pH.

Catalysis↗
PubMed2002

Determination of glucose oxidase immobilised as monolayer onto a flat surface.

An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.

Aspergillus niger↗
PubMed1997

Bioaffinity layering: a novel strategy for the immobilization of large quantities of glycoenzymes.

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae.

Biosensing Techniques↗
PubMed2003

Lab-on-a-chip for drug development.

Significant advances have been made in the development of micro-scale technologies for biomedical and drug discovery applications. The first generation of microfluidics-based analytical devices have been designed and are already functional. Microfluidic devices offer unique advantages in sample handling, reagent mixing, separation, and detection. We introduce and review microfluidic concepts, microconstruction techniques, and methods such as flow-injection analysis, electrokinesis, and cell manipulation. Advances in micro-device technology for proteomics, sample preconditioning, immunoassays, electrospray ionization mass spectrometry, and polymerase chain reaction are also reviewed.

Animals↗
PubMed1989

High-performance liquid chromatographic determination of some polar phospholipids in serum.

The experimental conditions have been optimized for high-performance liquid chromatographic determination of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) in serum. The phospholipids are separated on a silica gel column, using a mobile phase of acetonitrile-methanol-water (100:10:18, v/v), with ultraviolet photometric detection at 200 nm. The limit of detection was 0.2 micrograms (in 20 microliters) for natural phospholipids and 2.5 micrograms for synthetic phospholipids; the relative standard deviation was ca. 5%. An alternative detection is tensammetry at a mercury electrode, at a potential of -1.8 V, with an a.c. current frequency of 60 Hz and an amplitude of 20 mV. The tensammetric detection has an advantage in its independence of the structure of the phospholipids. In measurements without a column (flow-injection analysis), the tensammetric detection also yields a somewhat lower limit of detection than photometry (0.15 micrograms per 20 microliters), but this value increases more than ten times in chromatographic detection. The precision is poorer and is more susceptible to interferences. The method was applied to the determination of the above substances in the blood of obese children, as a function of physical stress and spa treatment. It was shown that physical exercise causes a decrease in the contents of PE and PC in the patients. On the other hand, the spa treatment has no pronounced effect on the phospholipid content in the blood.

Adolescent↗
PubMed2002

Spectrophotometric determination of diaminopyrimidines using benzoquinone.

The diaminopyrimidine derivatives trimethoprim (TMP), pyrimethamine (PMA) and 2,4-diaminopyrimidine (2,4-DAP) are found to react readily and efficiently in an aqueous solution with p-benzoquinone (p-BQ) to form a colored product with an absorption optimum wavelength of about 500 nm. The optimum reaction time, pH, temperature, solvent, and [p-BQ] are determined by separate trials. These conditions are confirmed by a MultiSimplex optimization method. The molar absorptivities of the TMP, PMA, and 2,4-DAP reaction products at 500 nm are 10,830, 10,650, and 9660 l mol(-1) cm(-1), respectively. TMP shows a linear range between 5 and 100 mg/l while PMA and 2,4-DAP exhibit linearity between 15 and 75 mg l(-1) and 5 and 30 mg l(-1), respectively. There is some specificity to this reaction; 2-aminopyrimidine does not react. Under the optimum conditions, sulfamethoxazole (SM) reacts rather poorly with a molar absorptivity of about 110 l mol(-) cm(-1). Using p-BQ, TMP in a pharmaceutical sample can be determined in the presence of SM using derivative spectrophotometry. The TMP-p-BQ reaction is adaptable for flow injection analysis.

Benzoquinones↗
PubMed1999

Capillary electrochemical enzyme immunoassay (CEEI) for phenobarbital in serum.

A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 microl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product (p-aminophenol) from the catalytic conversion of the substrate (p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 microg l(-1) (2.8 pmoles or 0.65 ng) and a range of 30-3000 microg l(-1). The assay could be used to determine the phenobarbital serum concentration in a 4 microl clinical serum sample without pretreatment.

Antibodies↗
PubMed1999

Urea determination using pH-enzyme electrode.

A pH-membrane electrode with n-tridodecylamine (TDDA) as the hydrogen-ion-selective ionophore was used for the construction of a potentiometric biosensor for urea determination. The electrode was enzymatically modified by covalent binding of urease molecules directly to the surface of the potentiometric membrane. Incorporation of the urea biosensor into simple double-channel flow injection analysis (FIA) system allows reproducible urea determination in a millimolar range of concentration. The utility and limitations of the presented biosensor-FIA system for analysis of various real samples has been investigated. The system can be useful for some biomedical and pharmaceutical applications such as analyses of urine, posthaemodialysis fluid and extracts from pharmaceutical ointments containing urea.

Biosensing Techniques↗
PubMed2000

Rapid, real-time sampling of R-84760 in blood by in vivo microdialysis with tandem mass spectrometry.

A new technique involving rapid sampling of R-84760 in real-time was achieved using a combination of microdialysis (MD) and tandem mass spectrometry (MS/MS). After collecting the analyte in real-time by MD and separating it by MS/MS, the ion intensities are adapted to the data without any subsequent chromatographic separation or flow injection analysis. The R-84760 concentration was obtained from the plateau part of the ion intensities or the corrected values using an internal standard, after immersing the MD probe into the dialysis solution containing the drug for a definite time. Since contamination of the ion source was prevented by using an organic solvent for the perfused solution, it was possible to establish a stable analysis method. For an MD membrane of length 4 mm, the R-84760 concentration in saline was linear over the range 5-541.1 ng/ml (r2 = 0.9997). Moreover, the R-84760 concentration in rat whole blood was linear over the range 24.9-1868.9 ng/ml (r2 = 0.9993). As this method allowed the measurement of free drug concentration in rat blood, the analysis was also able to provide data needed for determining the protein-binding ratio. The protein-binding ratio obtained from the calibration curve in saline and rat whole blood was 87-90%, which was close to the result obtained by another analysis method. The concentration profile of R-84760 in blood as obtained by the MD-MS/MS method correlated well with the concentration profile in plasma, which was simultaneously monitored by LC-MS/MS.

Analgesics↗
PubMed2000

Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.

Calibration↗
PubMed2000

Chemiluminescent choline biosensor using histidine-modified peroxidase immobilised on metal-chelate substituted beads and choline oxidase immobilised on anion-exchanger beads co-entrapped in a photocrosslinkable polymer.

A novel sensing layer design is presented based on the non-covalent immobilisation of enzymes on derivatized Sepharose beads subsequently entrapped in PVA-SbQ photopolymer. Two different modified Sepharose beads were used, IDA- and DEAE-Sepharose, for the immobilisation, respectively, of horseradish peroxidase (HRP) modified with histidine, and choline oxidase (Chx). The HRP-IDA-Sepharose-based sensing layer was used in a flow injection analysis chemiluminescent system as the basis of an H2O2 biosensor. It was shown that the pre-immobilisation on IDA-Sepharose beads enhanced the sensing layer stability and enabled the immobilisation of a larger amount of enzyme. A 1.8 mg charge of HRP-IDA-Sepharose beads in the sensing layer produced the most sensitive H2O2 biosensor. Such an analytical system exhibited very good performances, with a cycle time of 2 min and a detection limit of 15 pmol (detection ranging over four decades at least), and an unusual long operational stability of 200 measurements (CV, 3.5%). The HRP-IDA-Sepharose beads were then combined with Chx-DEAE-Sepharose. With this modified Sepharose-based biosensor the limit of detection for choline (S/N, 3) was equal to 0.5 pmol and the working range was 0.35 pmol-10 nmol. Moreover, the cycle time was only 2.5 min with the new sensing layer, and a long operational stability of 150 successive assays was found, with a variation coefficient of 2.6%.

Alcohol Oxidoreductases↗