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PubMed2003

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry.

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.

Adult↗
PubMed1993

Adhesion of parasitized red blood cells to cultured endothelial cells: a flow-based study of isolates from Gambian children with falciparum malaria.

Adhesion of parasitized red blood cells to vascular endothelium is thought to play an important role in the development of the ischaemic complications associated with severe falciparum malaria. Using a novel, flow-based assay, we have investigated the adhesion of parasitized red blood cells to formalin-fixed human umbilical vein endothelial cells (HUVEC), for isolates obtained from 32 Gambian subjects with mild or severe falciparum malaria. Red cells infected with wild strains of Plasmodium falciparum were able to adhere to HUVEC under physiologically relevant flow conditions, but the level of adhesion was highly variable, ranging from 1 to 688 adherent cells per mm2 of HUVEC. Within isolates, some adherent parasitized cells remained stationary, whilst other formed less stable interactions and rolled slowly over the cell surface. There was no significant difference in adhesion of parasitized cells between isolates obtained from mild or severe cases of malaria, although a subset of isolates did show very high levels of adhesion. The results suggest that there is not a simple relationship between the adhesion of parasitized cells to cultured endothelial cells (presumably via the receptor ICAM-1) and the clinical severity of the disease, although variation in microvascular adhesion in vivo may still be a determinant of ischaemic complications.

Cell Adhesion↗
PubMed1991

Operation of ion-selective electrode detectors in the sub-Nernstian/linear response range: application to flow-injection/enzymatic determination of L-glutamine in bioreactor media.

A novel approach for eliminating positive errors from endogenous ionic interferences when using ion-selective electrodes as detectors in flow-injection enzyme-based blosensing configurations is described. The method involves using a high background level of interfering ions in the sample diluent/carrier stream to convert the normally logarithmic potentiometric sensor into a linear detector over a given concentration range of primary ions. A split-stream single-detector arrangement provides a convenient means to compensate for varying levels of background interferent ions in the injected samples. One portion of the split stream passes directly to the ion-electrode detector, yielding a signal linearly related to the concentration of endogenous primary ions in the sample. The second portion of the split sample is delayed while passing through an immobilized enzyme that generates electrode detectable primary ions in proportion to the concentration of the substrate analyte in the sample. Two linear equations with two unknowns describe the twin potentiometric responses observed. The concept is demonstrated by the accurate determination of L-glutamine in hybridoma bioreactor media via the use of an ammonium-ion-selective membrane electrode detector and immobilized glutaminase enzyme.

Biosensing Techniques↗
PubMed1991

Liposome flow injection immunoassay: model calculations of competitive immunoreactions involving univalent and multivalent ligands.

The use of liposomes as detectable reagents in solid-phase immunoassays has been explored in a flow injection immunoanalysis (FIIA) system. Model calculations are presented for FIIA based on the competitive binding of univalent analyte and multivalent liposomes to immobilized antibodies. Parameters such as binding constants, concentrations of liposomes and antibody, and steric hindrance are considered for their relative effects on detectable liposome signal response to analyte concentrations. Qualitative comparisons of the model with the experimental data are made.

Flow Injection Analysis↗
PubMed1991

Cobalt(II)-catalyzed chemiluminescence in a dioctadecyldimethylammonium chloride bilayer membranous medium for the flow injection determination of phenylpyruvic acid.

A novel chemiluminescence system is described for the flow injection determination of phenylpyruvic acid (PPA). The presence of both ordered surfactant molecular assemblies and a metal ion catalyst in the system is essential for the phosphorescence of benzaldehyde (emitter) produced by the aerobic oxidation of PPA in alkaline solution. Dioctadecyldimethylammonium chloride bilayer aggregates and cobalt (II) allows PPA to be selectively determined down to 1 X 10(-7) M. The linearity is 2 orders of magnitude with a relative standard deviation 3.1% (n = 10) for 1 X 10(-6) M PPA. Of 28 other species (1 X 10(-3) M) tested, only 4-hydroxymanderic acid, 4-hydroxyphenylpyruvic acid, 2,5-dihydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylpyruvic acid provided signals 2-13 times more intense than that for 1 X 10(-6) M PPA. PPA present at 10(-3) - 10(-2) M levels in urine from patients with phenylketonuria can be determined with no special sample pretreatment by using this CL procedure. The mechanistic study of the present luminescent reaction are also undertaken in detail. The bilayer aggregates were found to contribute favorably both to the production of key intermediates and to the efficient phosphorescence emission.

Adult↗
PubMed1991

Fundamentals of sinusoidal flow sequential injection spectrophotometry.

A rational design of the sequential injection analyzer is based on description of the mutual penetration of sample and reagent zones, which are sequentially stacked into a tubular conduit and then injected into a reactor and transported toward the detector by means of a carrier stream, flowing at a preprogrammed rate. Variables governing zone penetration have been identified by a series of dispersion experiments, for sequential injection of two and three zones, thus outlining the conditions for performing single- and double-reagent-based assays. A parallel is drawn between conventional flow injection and corresponding sequential injection colorimetric determinations of chloride and phosphate, with the aim of suggesting guidelines for the development of spectrophotometric- and fluorescence-based sequential injection methods.

Flow Injection Analysis↗
PubMed2001

Voltammetric determination of L-cysteine at conductive diamond electrodes.

Boron-doped diamond (BDD) electrodes were used to examine L-cysteine (CySH) oxidation in alkaline media. The results of the voltammetric and polarization measurements showed that at BDD electrodes the overall CySH oxidation reaction is controlled by the initial electrochemical step, i.e., the oxidation of the CyS- electroactive species. The same conclusion was supported by the results of a study of pH effects. Conversely, at glassy carbon (GC) electrodes, the same reaction is controlled by the desorption of the reaction products. These results account for the poor response for CySH determination at GC compared to BDD. It was found that BDD exhibits excellent behavior for CySH determination, clearly outperforming GC. The results demonstrate that measurement of the peak current for CySH oxidation can be used as a basis for simple method for determining CySH in the micromolar concentration range by the use of BDD electrodes.

Cysteine↗
PubMed1993

Detection of liquid injection using an atmospheric pressure ionization radiofrequency plasma source.

An atmospheric pressure rf plasma source which operates in a variety of different buffer gases has been developed as an ionization method for organic samples introduced by liquid injection into atmospheric pressure ionization mass spectrometry (API/MS). The rf source can operate in He at < 1 W of load power at 165 kHz. It can also be sustained in Ar, N2, air, and CO2 at a load power of < 15 W. In most cases studied, the protonated molecule, MH+, is observed with little or no fragmentation even under the relatively high current conditions of the discharge. However, using increasingly higher acceleration voltages between the skimmers in the differentially pumped region between atmospheric pressure and high vacuum, one can induce fragmentation via collision-induced dissociation. This can be assisted in these experiments via the use of a heavy buffer gas. The detection limits achieved for rf/API plasma detection are typically in the low femtomole region for small organic molecules including neurotransmitters, PTH-amino acids, steroids, drugs, pesticides, and explosives. The detection can be performed with quantitation over at least 4 orders of magnitude.

Air Ionization↗
PubMed1993

Collisional fragmentation of glycopeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests.

Mass spectrometric methods of glycopeptide-specific detection in liquid chromatography/electrospray mass spectrometry (LC/ESMS) of glycoprotein digests are explored using a variety of glycopeptide models and then applied to soluble complement receptor type I, a 240-kDa glycoprotein containing 25 potential sites of N-glycosylation. The most specific method, requiring a triple quadrupole, involves monitoring of sugar oxonium fragment ions during precursor-ion scan ESMS/MS. Signals derived from nonglycosylated peptides are virtually eliminated, resulting in a total-ion current chromatographic trace of only the glycopeptides present in the digest. The corresponding mass spectra yield molecular weight and glycopeptide microheterogeneity information. An alternative and complementary approach that we term collisional-excitation scanning also involves fragmentation of glycopeptides to sugar oxonium ion fragments but does not involve any mass-selection process, permitting the experiment to be performed on a single quadrupole instrument. The resulting total ion chromatogram is similar to the UV chromatogram (215 nm), but a selected-ion chromatogram for carbohydrate-specific ions such as the N-acetylhexosamine oxonium ion (m/z 204) produces a glycopeptide-specific trace. Although there can sometimes be peptide interferences in the spectra of the indicated glycopeptide-containing chromatographic peaks, this latter approach permits peptide mapping to be performed on the same data set that also indicates the location of glycopeptides in the chromatogram. Both methods are suitable for detection of glycopeptides with all common classes of oligosaccharides in either N- or O-linkage to the peptide.

Carbohydrate Sequence↗
PubMed1993

Noncompetitive flow injection immunoassay for a hapten, alpha-(difluoromethyl)ornithine.

A noncompetitive flow injection immunoassay method has been developed to assay small haptens. In this assay the sample containing the hapten is incubated with excess enzyme-labeled monovalent antibody for a brief period. The excess antibody is then separated from the bound antibody by eluting through an antigen-immobilized immunoaffinity column. The enzyme label of the eluting antibody-hapten complex is fluorometrically detected. The applicability of the method is demonstrated by assaying alpha-(difluoromethyl)ornithine (DFMO), an anticancer drug in human plasma samples. The assay is sensitive enough to detect 200 amol of DFMO. Interferences from other similar endogenous amines have been eliminated by selective immunoaffinity purification of the antibodies.

Animals↗
PubMed1993

Specific flow injection sandwich binding assay for IgG using protein A and a fusion protein.

A sandwich-type flow-injection binding assay for quantitation of various IgG's was developed. The assay is based on the pseudoimmunological reaction between protein A from Staphylococcus aureus and immunoglobulin G from different species. Protein A immobilized on a solid support and a fusion protein of protein A and beta-galactosidase from Escherichia coli are used for detection. The fusion protein is produced with a temperature-inducible recombinant E. coli strain. A sandwich is formed by subsequent injection of IgG and fusion protein into the buffer stream flowing through the immobilized protein A column. The amount of enzyme activity bound is proportional to the amount of IgG bound and is measured by pumping a lactose solution as substrate for beta-galactosidase through the protein A column. Lactose is converted to glucose and galactose. The detector is an enzyme thermistor that measures the heat evolved in the enzymatic conversion of glucose by coimmobilized glucose oxidase and catalase. The assay takes 16 min at a flow rate of 0.6 mL min-1 with a lower detection limit of 33 pmol per injection of rabbit IgG. The precision of replicate measurements has a standard deviation of 4-5%, and the column can be used for more than 50 cycles.

Animals↗
PubMed1993

Jet ring cell: a tool for flow injection spectroscopy and microscopy on a renewable solid support.

A new flow cell design for spectroscopic measurements of suspensions, the jet ring cell, is introduced. This cell exploits radial flow through a narrow ring-shaped gap to retain suspended particles within the detection region. This ring constitutes a detection volume of well-defined area from which the trapped particles can be instantaneously removed at will. The bed of particles thus forms a renewable surface, which can be probed by reflectance, fluorescence, or chemiluminescence using a microscope or optical fiber. This device should prove useful for microscopic study of cells, for automated immunoassays, and for preconcentration of analytes on sorbents with in situ spectroscopic detection. In conjunction with a fiber optic detection system, the jet ring cell becomes a component of a renewable chemical sensor system.

Animals↗
PubMed1994

Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum.

Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).

Alcohol Oxidoreductases↗
PubMed1994

Flow injection renewable surface immunoassay: a new approach to immunoanalysis with fluorescence detection.

This paper introduces a new methodology of carrying out heterogeneous immunoassays automatically, using a flow injection technique on a renewable surface. Flow injection renewable surface immunoassay (FIRSI) relies on the use of a minute amount of beads to form a reactive surface, which is interrogated by fluorescence spectrometry. Following the assay, on-line regeneration normally used in flow based immunoassays is avoided by fluidically removing the spent reactive surface and replacing it with a new layer of beads. This allows the monitoring of antibody-antigen binding at its early stages, dramatically increases the sampling frequency of a serial assay, and eliminates the problems associated with a decrease in surface reactivity caused by repetitive use. A model system utilizing anti-mouse IgG1-coated beads and mouse IgG1 protein is used to characterize the method with respect to reproducibility, flow rate, contact time, and amount of beads.

Animals↗
PubMed2001

Needle-type dual microsensor for the simultaneous monitoring of glucose and insulin.

A miniature needle-type sensor suitable for the simultaneous amperometric monitoring of glucose and insulin is described. The integrated microsensor consists of dual (biologically and chemically) modified carbon-paste working electrodes inserted into a 14-guage needle. The glucose probe is based on the biocatalytic action of glucose oxidase, and the insulin one relies on the electrocatalytic activity of ruthenium oxide. The analytical performance of the dual sensor is assessed under flow injection conditions. The needle dual detector exhibits a very rapid response to dynamic changes in the concentrations of glucose and insulin. No apparent cross reactivity is observed in mixtures containing millimolar glucose levels and nanomolar insulin concentrations. The response is highly linear (to at least 1000 nM insulin and 14 mM glucose) and reproducible (RSD = 2.6-4.1%). The combination microsensor holds great promise for real-time measurements of the insulin/glucose ratio and for improved management of diabetes.

Biosensing Techniques↗