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Biomedical subjects

M R Smyth

Publications and source records attributed to M R Smyth.

At least 37 records · Page 2Linked to original sources
PubMed1996

Oriented immobilization of antibodies and its applications in immunoassays and immunosensors.

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Antibodies↗
PubMed1996

Differential-pulse voltammetric determination of clenbuterol in bovine urine using a Nafion-modified carbon paste electrode.

A method has been developed for the determination of clenbuterol in bovine urine using differential-pulse voltammetry (DPV), based on the electrochemical behaviour of clenbuterol at a Nafion-modified carbon paste electrode (CPE). Clenbuterol is irreversibly oxidized at high positive potentials, its irreversibility being due to a chemical follow-up reaction which results in a product showing quasi-reversible electrochemical behaviour at much lower potentials. It is the oxidation peak of this product, arising in acidic media at 0.42 V, which was analysed using DPV, again following the accumulation of clenbuterol at the Nafion-modified CPE. Electrode renewal was achieved by holding the potential at -0.6 V for 120 s in 0.1 mol l-1 NaOH. The determination of clenbuterol in the presence of interfering compounds present in bovine urine samples was then carried out after a two-step clean-up of the urine involving liquid-liquid extraction followed by a mixed-mode solid-phase extraction procedure. This allowed clenbuterol to be detected down to a level of 1.02 x 10(-9) mol l-1 in bovine urine extracts.

Adrenergic beta-Agonists↗
PubMed1996

Interspecies differences in coumarin metabolism in liver microsomes examined by capillary electrophoresis.

1. A simple, rapid method was developed for studying xenobiotic metabolism by cytochrome P450 in liver microsome preparations. Capillary electrophoresis was used to separate the metabolite from the metabolic mixture. 2. Coumarin is metabolized to 7-hydroxycoumarin by a cytochrome P450 isoenzyme. Human, bovine, gerbil, mouse (Schofield, CO1), rat, rabbit, porcine, and cynomologus monkey microsomal preparations were investigated for coumarin metabolism by determining the content of 7-hydroxycoumarin present after metabolism. 3. Separation of 7-hydroxycoumarin from the reaction mixture was carried out in 50 mM phosphate buffer, pH 6.8, on a fused silica capillary at 25 degrees C and 15 kV. The metabolic matrix consisted of an NADPH regeneration system, 205.5 mu M coumarin, and the microsomal preparation. Standard curves were prepared in the microsomal preparation and the limit of quantification was 6.17 mu M, with a linear range from 0 to 308.5 mu M. 4. The reaction was initiated by the addition of the microsomes. An aliquot of the reaction mixture was removed at specific timed intervals over 2 h and injected directly onto a capillary electrophoresis column and the concentration of 7-hydroxycoumarin determined. The metabolism of coumarin to 7-hydroxycoumarin is greatest in human and monkey microsomes.

Animals↗
PubMed1995

Determination of free and total 7-hydroxycoumarin in urine and serum by capillary electrophoresis.

A new method for the rapid determination of 7-hydroxycoumarin, the predominant metabolite of coumarin in humans, was developed for analysis in urine and serum, based on separation by capillary electrophoresis, with UV detection at 210 nm. The linear detection range for 7-hydroxycoumarin was 0-50 micrograms/ml while the limit of quantitation was 1 microgram/ml. An internal standard, 3-(alpha-acetonylbenzyl)-4-hydroxycoumarin, was utilised for the determination of free 7-hydroxycoumarin, but it was found not to be suitable in the analysis of total 7-hydroxycoumarin present. Urine from two volunteers, who had been administered coumarin, was analysed by both capillary electrophoresis and by HPLC. The results from the two methods were compared and contrasted. The CE method was found to decrease the analysis time in comparison to HPLC analysis, with results available after 1.5 min as compared to 12 min with HPLC. There was no statistical difference between the results determined by either method.

Coumarins↗
PubMed1995

Electrochemical behaviour of clenbuterol at Nafion-modified carbon-paste electrodes.

A detailed study of the electrochemistry of clenbuterol at bare carbon-paste electrodes (CPEs) has been carried out. Results showed that clenbuterol undergoes an ECE process. This compound is irreversibly oxidised at high potentials, resulting in the formation of a product which demonstrates quasi-reversible electrochemical behaviour at less positive potentials. The amount of this chemical product formed is very pH-dependent. Investigations into the electrochemical behaviour of clenbuterol at Nafion-modified CPEs were also made. The use of a thin Nafion film cast over the CPE resulted in a large increase in peak current over bare electrodes. Linear accumulation occurred with time, the linear range increasing with decreasing concentration. This allowed the detection of low concentrations of clenbuterol. Diffusion proved to be the rate-controlling process of clenbuterol through the Nafion membrane.

Carbon↗
PubMed1995

The use of differential measurements with a glucose biosensor for interference compensation during glucose determinations by flow injection analysis.

A novel detection system for the determination of glucose in the presence of clinically important interferents, based on the use of dual sensors and flow-injection analysis (FIA), is described. The normalisation methodology involves measurement of the interference signal at a reference sensor; this signal can then be subtracted from the glucose sensor signal (post-run) to give a corrected measurement of the glucose concentration. The detection system consists of a thin layer with dual glassy carbon working electrodes. One electrode was surface modified to act as a glucose biosensor by immobilisation of glucose oxidase (GOx) (from Aspergillus niger) with 1% glutaraldehyde and bovine serum albumin. The second electrode (glucose oxidase omitted) was utilised to measure the interference signal responding only to electroactive species present in the injected sample. A computer controlled multichannel potentiostat was used for potential application and current monitoring duties. The sensor responses were saved in ASCII format to facilitate post-run analysis in Microsoft Excel. Cyclic voltammetry (CV) was utilised to investigate the manner in which the interference signal contributed to the total signal obtained at the biosensor in the presence of glucose. The kinetics parameters Imax and the apparent Michaelis-Menten constant, K'm, were calculated for the sensor operating under flow-injection conditions.

Acetaminophen↗
PubMed1994

Antibody-based approaches to coumarin analysis.

7-Hydroxycoumarin (7-HC) was chemically conjugated by diazo coupling to carrier proteins such as bovine serum albumin (BSA), thyroglobulin and ovalbumin. These conjugates were characterised by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Rabbits were immunised using the 7-HC-BSA conjugate. The highest antibody titre achieved was 1:10,000, as determined by competitive enzyme-linked immunosorbent assay (ELISA). The resulting antibodies were purified by ammonium sulphate precipitation, followed by protein A affinity chromatography. Their purity was assessed by SDS-PAGE and HPLC. These antibodies have been used in the development of a competitive ELISA, an amperometric biosensor and an electrochemical immunoassay. Both the ELISA and amperometric biosensor have been successfully applied to the analysis of 7-HC and its glucuronide conjugate in human urine samples. Each of these antibody-based methods provides a novel approach to the analysis of the main metabolites of coumarin.

Animals↗
PubMed1994

Thermostabilized chemical derivatives of horseradish peroxidase.

Horseradish peroxidase finds a variety of uses in analysis, immunology, organic synthesis, and biosensors. Although moderately stable, its applicability to biosensors and other fields would be greatly enhanced if it could be made yet more stable. Appropriate chemical modification can substantially stabilize enzymes. Here we describe the use of bis-imidates and of bis-succinimides to modify free amino groups of commercial horseradish peroxidase under mild conditions of pH and temperature. Imidates yielded a marginal stabilization. Some of the succinimide derivatives, however, are much more thermostable than the native enzyme. Apparent half-lives indicate stabilizations of 6- to 23-fold, depending on the bis-succinimide used. These modifications preserve the carbohydrate side chains for subsequent reaction or immobilization.

Biosensing Techniques↗
PubMed1994

Application of a Nafion-modified carbon paste electrode for the absorptive stripping voltammetric determination of fenoterol in pharmaceutical preparations and biological fluids.

The preconcentration of fenoterol on a Nafion-modified carbon paste electrode and its subsequent determination using differential pulse voltammetry is described. The effect of pH and percentage Nafion concentration on the accumulation behaviour of fenoterol was studied, and accumulation curves, calibration graphs and reproducibility studies at two different Nafion concentrations have been carried out in the range 2.5 x 10(-8)-5.0 x 10(-7) M fenoterol. A limit of detection in aqueous solutions, calculated using a signal-to-noise ratio (S/N) of 3, was 9.0 x 10(-9) M. Application of the electrode to pharmaceutical preparations, without sample pretreatment, resulted in acceptable deviation from the stated concentration (RSD = +/- 3.81%, n = 4). For more complex matrices, a suitable extraction procedure was developed, resulting in recoveries of > 90% (urine) and > 75% (serum).

Adsorption↗
PubMed1994

Determination of proanthocyanidins and catechins in beer and barley by high-performance liquid chromatography with dual-electrode electrochemical detection.

A method was developed to determine the flavanols prodelphinidin B3, procyanidin B3, (+)-catechin, and (-)-epicatechin by high-performance liquid chromatography, using dual-channel electrochemical detection. This method was especially suited to the direct analysis of beer samples and to analysis of acetone extracts of barley samples, and was capable of determining proanthocyanidins and catechins at levels of 0.1-5.0 mg l-1. The use of dual-channel electrochemical detection also enabled unambiguous peak identification by measurement of collection efficiencies. This method offered improved sensitivity and selectivity compared with ultraviolet detection, and sample preparation procedures were greatly simplified. The method was applied to the comparison of stabilized and unstabilized lagers, and to the analysis of different barley varieties grown in Ireland.

Anthocyanins↗
PubMed1994

Matrix solid-phase dispersion as a multiresidue extraction technique for beta-agonists in bovine liver tissue.

A procedure for the extraction of salbutamol including conjugated forms of the drug, from liver samples, is described. It combines matrix solid-phase dispersion with radioimmunoassay for the measurement of salbutamol residues at the sub-ppb level. Inter- and intra-assay validation, carried out on fortified liver samples, show good recoveries over the range 1-5 ppb of salbutamol. An enzyme hydrolysis procedure was optimized for the deconjugation of incurred residue. The developed procedure is shown to be suitable for the extraction and determination of other beta-agonists such as clenbuterol, mabuterol, terbutaline, and cimaterol at residue levels of less than 1 ng g-1.

Adrenergic beta-Agonists↗
PubMed1994

Multi-residue analysis for beta-agonists in urine and liver samples using mixed phase columns with determination by radioimmunoassay.

A method is described for the extraction of four beta-agonists, clenbuterol, salbutamol, mabuterol and terbutaline from bovine urine and liver samples using radioimmunoassay (RIA) as the method of determination. Following enzymic digestion of the liver samples using protease enzyme, the digest is centrifuged and the harvested supernatant is saturated with sodium chloride and adjusted to pH 11.0. An ethyl acetate-propan-2-ol mixture is used to extract the beta-agonists from the liver digest. The samples of urine and liver extracts are adjusted to pH 6.0 and applied to mixed phase (XtrackT) columns. The column is washed with water and methanol and the beta-agonists are eluted with methanol containing 2% ammonia. After evaporation of the eluting solvent and reconstitution in ethanol the beta-agonist residues are determined by RIA, with standard graphs prepared in residue-free sample extract. The procedure has been validated for clenbuterol, salbutamol, mabuterol and terbutaline. The mean recovery of the beta-agonists from urine and liver is > 75% and > 85%, respectively. The detection limit is 0.13 ng ml-1 and 0.46 ng g-1 of clenbuterol in urine and liver, respectively. The high recoveries attained for both types of beta-agonists are a result of an efficient liquid-liquid extraction step coupled with a selective mixed solid-phase extraction procedure.

Adrenergic beta-Agonists↗
PubMed1994

Horseradish peroxidase: the analyst's friend.

HRP is a calcium-containing, extensively glycosylated, stable haemoprotein possessing four disulphide bridges and weighing 44 kDa. It is an oxidoreductase which can use a wide variety of hydrogen donors to reduce hydrogen peroxide. This gives rise to a range of colorimetric, fluorimetric, chemiluminescent and electrochemical assays for HRP activity. HRP is very widely used as an indicator in immunoassays, non-isotopic DNA probes, cytochemistry, bi-enzyme systems and biosensors. This is owing to its stability, high catalytic rates, ease of conjugation to other molecules and wide choice of assays of activity.

Biosensing Techniques↗
PubMed1993

Determination of peptides by capillary electrophoresis-electrochemical detection using on-column Cu(II) complexation.

A Cu(II)-coated capillary has been developed for the determination of peptides by capillary electrophoresis with electrochemical detection. Capillaries were prepared by forcing a solution 48 microM in CuSO4, 120 microM in tartaric acid, 2.4 mM in NaOH and 120 microM in KI through them for 25 min; the resulting capillaries are stable for at least 12 h. Under alkaline conditions, peptides complex with Cu(II) present on the walls of the capillary to form Cu(II)-peptide complexes which can be detected oxidatively at a carbon fiber electrode. Di-, tri-, tetra- and pentaglycine were determined with a detection limit of 7 x 10(-7) M for triglycine. N-Terminal-blocked peptides can also be determined via this method. This system is more sensitive than direct detection of peptides by UV at 210 nm and exhibits higher selectivity than commonly employed derivatization procedures based on reactions with a primary amine functionality.

Amino Acid Sequence↗
PubMed1993

Voltammetric study of salbutamol and application to its determination in a tablet dosage form and dissolution profiles for the dosage form.

The oxidative voltammetric behaviour of salbutamol at a glassy carbon electrode surface has been studied using cyclic voltammetry and differential pulse voltammetry. Oxidation of the drug was effected in a single irreversible, adsorption-controlled step in phosphate buffer. The process was found to be dependent on the ionic strength and the pH of the supporting electrolyte. The response was evaluated with respect to accumulation time, scan rate and other variables. Using differential pulse voltammetry following electrochemical pretreatment of the electrode surface, the drug yielded a well-defined voltammetric response in phosphate buffer, pH 5.0, at +0.75 V (vs SCE). This process could be used to determine salbutamol concentrations in the range 8 x 10(-7) M to 8 x 10(-5) M with a detection limit of 2 x 10(-7) M. The method was applied, without any interferences from the excipients, to the determination of the drug in a tablet dosage form and in drug dissolution studies. The absolute recovery for salbutamol was greater than 95% at the concentration levels studied, and reproducible voltammetric signals were obtained with a relative standard deviation of 2.4% for n = 7 at a concentration level of 8 x 10(-5) M.

Adsorption↗
PubMed1993

Phase-selective AC adsorptive stripping voltammetric assay for aminopterin and 10-Edam in human serum.

Aminopterin was studied as a model compound for its analogues which maintain the pteridine ring in their structure. Its adsorptive behaviour on mercury was studied and the DC adsorptive stripping and phase-selective AC adsorptive stripping conditions were optimized. 10-Edam, an aminopterin analogue, was studied and shown to behave similarly to aminopterin. Phase-selective AC voltammetry provided the best signal and gave a detection limit of 4 x 10(-12) M aminopterin in aqueous solution employing an accumulation time of 10 min. The optimized method was applied to the analysis of both aminopterin and 10-Edam respectively in human serum. After extraction with a C18 reversed-phase cartridge the detection limit of the method was 1 x 10(-8) M aminopterin and the overall assay percentage recovery was 73.5% (n = 5) at a concentration of 5 x 10(-7) M aminopterin in serum. The analysis of 10-Edam at the same concentration in serum yielded the higher percentage recovery of 94.46% (n = 5) following the same procedure.

Adsorption↗
PubMed1993

Differential pulse voltammetric determination of 7-hydroxycoumarin in human urine.

The electrochemical behaviour of 7-OH-coumarin at the bare glassy carbon electrode has been studied using differential pulse voltammetry, and based on anodic detection of this metabolite at 0.66 V (vs SCE) using DC amperometry, a method has been developed for the determination of 7-OH-coumarin levels in urine samples, and a pharmacokinetic profile established.

Electrodes↗