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PubMedDate not supplied

Determination of nickel in biological samples spectrophotometrically involving complexation reaction with some thiazolylazo compounds.

Three new heterocyclic azo compounds, 5(2-benzothiazolylazo)2,2 biphenyldiol (I), 5-(2-benzothiazolylazo)-8-hydroxyquinolene (II) and 4-(2-benzothiazo-lylazo)3-hydroxy-2-naphthoic acid (III) were synthesized. The formation constant of the reagents in 30% (v/v) ethanol and reactions with various metal ions were studied. These reagents (I-III) reacts with nickel ion to form (2:1) colored complex with an absorption band at 604, 635 and 643 nm. The apparent stability constants and the optimum conditions for complete color development were investigated. Beer's law is obeyed over the concentration ranges of 0.05-3.50, 0.05-4.00 and 0.05-3.10 micrograms ml-1. For more accurate analyses, Ringbom optimum concentration ranges were found to be 0.20-3.25, 0.20-3.80 and 0.20-2.90 micrograms ml-1 using reagents I, II and III, respectively. The apparent molar absorptivity and Sandell sensitivity were also calculated. The interference of various foreign ions on the determination of nickel was investigated. The proposed method has been successfully applied to the determination of nickel substrates in various biological samples.

Animals↗
PubMed1990

Simultaneous determination of pantothenic acid and hopantenic acid in biological samples and natural products by gas chromatography-mass fragmentography.

A method for the simultaneous determination of pantothenic acid and hopantenic acid in plasma samples was developed using gas chromatography-mass spectrometry with multiple ion detection. Plasma samples were directly purified without deproteinization on an ion-exchange resin, and the eluate was extracted with ethyl acetate under acidic conditions. The organic layer was evaporated to dryness under a stream of nitrogen, and the residue was dissolved in an internal standard solution. Pantothenic and hopantenic acids were converted into their trimethylsilyl derivatives by treating with bis(trimethylsilyl)trifluoroacetamide. Aliquots of this solution were injected into the gas chromatograph-mass spectrometer, which was equipped with a wide-bore fused-silica column (DB-17) and analysed by the multiple ion detection method. The detection limits for pantothenic acid and hopantenic acid in plasma were 1 ng/ml each at a signal-to-noise ratio of 5. This method was applied to a study of the assay of pantothenic acid and hopantenic acid in biological samples and natural products.

Animals↗
PubMed1992

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.

Animals↗
PubMed1993

Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.

The development of immunoassays and immunoaffinity chromatography methods for determination of carcinogen-DNA adducts and carcinogen-modified DNA samples rests upon eliciting and characterizing polyclonal and monoclonal antisera against these haptens. The use of such antisera has widespread application in investigating chronic carcinogen administration in animal models and in monitoring human tissues for evidence of carcinogen exposure. Radioimmunoassays and enzyme-linked immunosorbent assays developed with carcinogen-DNA adduct antisera are exceedingly sensitive, measuring 1 adduct in 10(8) nucleotides. Not only can DNA damage be quantified directly by immunoassay, but the antisera have also been used to isolate DNA adducts of a particular chemical class by immunoaffinity chromatography before application of more chemically-specific end-points. Both of these methodological approaches have made seminal contributions to the newly-emerging field of molecular epidemiology. This chapter will focus on methods for preparing immunogens, the establishment of immunoassays, characterization of antisera and specific problems encountered with biological samples in addition, the use of immunoaffinity chromatography for preparative concentration of DNA adducts of a particular class will be included.

Animals↗
PubMed2003

A method for detecting O-glycanase in biological samples using a combination of MALDI-TOF mass spectrometry and time-resolved fluorimetry.

O-Glycans (mucin type oligosaccharides) are ubiquitously found in various glycoproteins such as mucin-type glycoproteins on the surface of various digestive organs. In the present paper, we propose a method for detecting O-glycanase which catalyzes the hydrolysis of the O-glycan linkage between oligosaccharides and serine/threonine residues of mucin-type glycoprotein. As the substrate for O-glycanases, we chose glycopeptides containing O-glycans derived from bovine fetuin. The present method is divided into two parts. At the initial stage, the presence of O-glycanase was confirmed by observing characteristic ions due to O-glycans and peptides released from the glycopeptide by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the second step, europium labeled O-glycosylated peptide permits more detailed analysis such as enzyme kinetics. We demonstrated the usefulness of the present method using O-glycanase (Streptococcus pneumoniae) as model enzyme. The present approach can easily confirm the presence of O-glycanase by detecting both deglycosylated peptides and O-glycans, even if contaminating peptides or glycosidases are present in crude biological samples.

Amino Acid Sequence↗
PubMedDate not supplied

Differential pulse polarographic determination of lead in standard alloys and biological samples after separation and preconcentration with PAN.

A highly selective, sensitive, rapid and economical differential pulse polarographic method has been developed for the determination of trace amount of lead in various samples after adsorption of its 1-(2-pyridylazo)-2-naphthol (PAN) on naphthalene in the pH range of 8.4 - 11.5. After filtration, the solid mass is shaken with 9.0 ml of 1 M hydrochloric acid and lead is determined by differential pulse polarography (DPP). Lead can alternatively be quantitatively adsorbed on [1-(2-pyridylazo)-2-naphthol]-naphthalene adsorbent packed in a column and determined similarly. The detection limit is 0.1 microg/ml (signal to noise ratio = 2) and the linearity is maintained in the concentration range 0.3 - 300 microg/ml with a correlation coefficient of 0.9996 and relative standard deviation of +/- 1.1%. Characterization of the electroactive process included an examination of the degree of reversibility. Various parameters such as the effect of pH, volume of aqueous phase and interference of a number of metal ions on the determination of lead has been studied in detail to optimize the conditions for determination in alloys and biological samples.

Alloys↗
PubMed2005

Simultaneous detection of [metal(II)-tpen]2+ as kinetically inert cationic complexes using pre-capillary derivatization electrophoresis: an application to biological samples.

A high resolution of doubly charged first row transition (Fe, Cu, Zn, Ni, Co, Mn) and heavy metal (Pb, Cd, Hg) ions was achieved in capillary electrophoresis (CE) with high sensitivity (sub-micromol dm(-3) level), using NN,N'N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) as a pre-capillary derivatizing agent. The non-charged reagent, TPEN, was applied to capillary zone electrophoresis (CZE) for the first time. Since complete spatial separation between the complexes and the ligand was carried out in a carrier buffer, which was free of TPEN, kinetic inertness of metal complexes was necessary for the detection in this pre-capillary method. All the nine listed metal complexes were detected: Ca(2+), Mg(2+), Al(3+), Fe(3+), and Co(3+) complexes were undetectable. This, interestingly, suggests that those nine cations form kinetically inert tpen complexes without strong charge-charge interactions between the metal ion and the ligand. It is expected that the hard-soft-acid-base (HSAB) principle governed the kinetics selectivity. With respect to the electrophoretic behavior, the addition of chloride ion and methanol to the carrier significantly improved the resolution. This is due to the formation of ternary complexes or ion aggregates and the solvation effect, respectively. These effects provided a satisfactory baseline resolution among the nine metal ions. An application to biological samples was demonstrated. Some metal ions in human serum and urine were successfully detected in a simple process without the need for deproteinization using a non-coated fused-silica capillary because of the differenciation in the direction of migration between organic matter and complexes.

Buffers↗
PubMed1990

Column chromatographic pre-concentration of iron(III) in alloys and biological samples with 1-nitroso-2-naphthol-3,6-disulphonate and benzyldimethyltetradecylammonium-perchlorate adsorbent supported on naphthalene using atomic absorption spectrometry.

The solid ion-pair material produced from the reaction between benzyldimethyltetradecylammonium chloride (BDTA) and sodium perchlorate on naphthalene provides the basis for a simple, rapid and selective technique for pre-concentrating iron from up to 500 ml of aqueous solution. Iron reacts with disodium 1-nitroso-2-naphthol-3,6-disulphonate (Nitroso-R salt) to form a water-soluble coloured chelate anion. The iron chelate anion forms a water-insoluble, stable iron-Nitroso-R-BDTA complex on naphthalene packed in a column. Trace amounts of iron are quantitatively retained on naphthalene in the pH range 3.5-7.5 and at a flow-rate of 1-2 ml min-1. The solid mass is dissolved out from the column with 5 ml of N,N-dimethylformamide and iron is determined by means of an atomic absorption spectrometer at 248 nm. The calibration graph is linear for concentrations of iron over the range of 0.5-20 micrograms in 5 ml of final solution. The standard deviation and relative standard deviation were calculated. The detection limit of the method was 0.0196 micrograms ml-1 of iron. The sensitivity for 1% absorption was 0.072 microgram ml-1 (0.165 microgram ml-1 by direct atomic absorption spectrometry of aqueous solution). The proposed method was applied to the determination of iron in standard alloys and biological samples.

Adsorption↗
PubMed1998

Organotin compounds in water, sediment, and biological samples from the port of osaka, japan

Butyltin (Bts) and phenyltin compounds (Pts) were measured in water, sediment, plankton, and mussels collected from eight stations in the harbor area of the Port of Osaka in 1996, and were compared with those of Otsuchi Bay. The levels of tributyltin (TBT) compounds in all samples from the Port of Osaka were slightly high in marinas and mooring areas of small and medium-hull vessels. Of total Bts, the ratio of TBT was under 50% in water and was dominant in sediment, plankton, and mussels. Triphenyltin (TPT) compounds in water were not detected. TPT was present at lower levels than TBT in sediment, plankton, and mussels. Of total Pts, monophenyltin (MPT) compounds and diphenyltin (DPT) compounds represented a high proportion in sediment, while TPT was dominant in plankton and mussels. The concentrations of TBT in water from the Port of Osaka were lower than those from Otsuchi Bay, however TBT concentrations in sediment, plankton, and mussels from the Port of Osaka were higher than those from Otsuchi Bay. The difference of TBT concentration among stations in the Port of Osaka was slight. In contrast, the levels of TBT in Otsuchi Bay were markedly higher at a station near the shipyard. Though TPT in water from the Port of Osaka was not detected, trace amounts of TPT was found in seawater from Otsuchi Bay. The levels and detected frequencies of TPT in sediment, plankton, and mussels from the Port of Osaka were lower than in those from Otsuchi Bay. The partition coefficients of Bts to sediment, plankton, and mussels from the Port of Osaka were higher than those of dibutyltin (DBT) compounds and MBT and those of TBT to plankton and mussels were higher than those in sediment. The partition coefficients of TBT to sediment and biological samples from the Port of Osaka were higher level than those in Otsuchi Bay.

Journal Article↗
PubMed2006

A 96-well screen filter plate for high-throughput biological sample preparation and LC-MS/MS analysis.

A novel 96-well screen filter plate (patent pending) has been invented to eliminate a time-consuming and labor-intensive step in preparation of in vivo study samples--to remove blood or plasma clots. These clots plug the pipet tips during a manual or automated sample-transfer step causing inaccurate pipetting or total pipetting failure. Traditionally, these blood and plasma clots are removed by picking them out manually one by one from each sample tube before any sample transfer can be made. This has significantly slowed the sample preparation process and has become a bottleneck for automated high-throughput sample preparation using robotic liquid handlers. Our novel screen filter plate was developed to solve this problem. The 96-well screen filter plate consists of 96 stainless steel wire-mesh screen tubes connected to the 96 openings of a top plate so that the screen filter plate can be readily inserted into a 96-well sample storage plate. Upon insertion, the blood and plasma clots are excluded from entering the screen tube while clear sample solutions flow freely into it. In this way, sample transfer can be easily completed by either manual or automated pipetting methods. In this report, three structurally diverse compounds were selected to evaluate and validate the use of the screen filter plate. The plasma samples of these compounds were transferred and processed in the presence and absence of the screen filter plate and then analyzed by LC-MS/MS methods. Our results showed a good agreement between the samples prepared with and without the screen filter plate, demonstrating the utility and efficiency of this novel device for preparation of blood and plasma samples. The device is simple, easy to use, and reusable. It can be employed for sample preparation of other biological fluids that contain floating particulates or aggregates.

Automation↗
PubMed2003

Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection.

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.

Bacillus subtilis↗
PubMedDate not supplied

Instrumental neutron activation analysis of biological samples.

The elemental compositions of 18 biological reference materials have been processed, for 14 stepped combinations of irradiation/decay/counting times, by the INAA Advance Prediction Computer Program. The 18 materials studied include 11 plant materials, 5 animal materials, and 2 other biological materials. Of these 18 materials, 14 are NBS Standard Reference Materials and four are IAEA reference materials. Overall, the results show that a mean of 52% of the input elements can be determined to a relative standard deviation of +/- 10% or better by reactor flux (thermal plus epithermal) INAA.

Animals↗
PubMed1995

Determination of aldehydes and other lipid peroxidation products in biological samples by gas chromatography-mass spectrometry.

The extremely broad spectrum of the biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that can quantitate all of the aldehydes formed in biological materials. The proposed method is based on the use of O-(2, 3, 4, 5, 6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) to form the O-pentafluorobenzyl-oxime (PFB-oxime) derivatives of 22 saturated and unsaturated aldehydes (C2-C12) including hexanal, 4-hydroxy-non-2-enal (HNE), and malondialdehyde (MDA), followed by trimethylsilylation of the hydroxyl group to trimethylsilyl (TMS) ethers. The PFB-oxime-TMS derivatives were analyzed by capillary column gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICIMS) with ammonia as reagent gas. Quantitation was achieved using benzaldehyde-ring-D5 as an internal standard in selected ion recording (SIR) mode. Standard curves were linear (r > 0.99) for all individual aldehydes. The detection limit was between 50 and 100 fmol per 1 microliter injected aldehyde. Recovery of all aldehydes from urine, plasma, and tissue homogenate was over 85%, except HNE, trans-2-octenal and trans-2,-cis-6-nonadienal from plasma and tissue sample, which were between 60 and 80%, suggesting these aldehydes may bind to protein and lipid components, especially to SH groups of proteins. The high sensitivity of this method allows the measurement of physiological aldehyde levels in biological samples. The products of aldehyde metabolism can also be measured by this assay.

Aldehydes↗
PubMed1981

Sample extraction detector for the liquid chromatographic determination of secoverine in biological samples.

A simple detection system for liquid chromatography based on post-column ion-pair extraction in a solvent-segmented stream is described. The fluorescent ion-pairing reagent 9,10-dimethoxyanthracene-2-sulphonate is added to the aqueous mobile phase prior to the column. An organic solvent, used for extraction of the ion pairs and for segmentation purposes, is added to the column effluent by gravity; about 70% of this phase is drawn through the detector cell using the siphon principle. The noise is significantly less than that of system with a pulsating peristaltic pump. The system has been used for the detection of the prospective drug secoverine. Under optimized conditions, band broadening due to the extraction detection system is sigma t = 7 sec. The detection limit of secoverine is ca. 20 pg, the repeatability in the nanogram range is 2% (relative standard deviation) (n = 6) and the linear range is at least 4 decades. The use of pre-column containing a 1--2-mm layer of stationary phase for clean-up and trace enrichment allows the direct injection of 200--1000 microliters of plasma or serum. In this way, secoverine can be determined in the parts per 10(12) range.

Chromatography, Liquid↗
PubMed1993

Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.

Scanning force microscopy allows imaging of biological molecules in their native state in buffer solution. To this end samples have to be fixed to a flat solid support so that they cannot be displaced by the scanning tip. Here we describe a method to achieve the covalent binding of biological samples to glass surfaces. Coverslips were chemically modified with the photoactivatable cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Samples are squeezed between derivatized coverslips and then cross-linked to the glass surface by irradiation with ultraviolet light. Such samples can be imaged repeatedly by the scanning force microscope without loss of image quality, whereas identical but not immobilized samples are pushed away by the stylus.

Azides↗
PubMed1998

Quantitative elemental X-ray imaging of frozen-hydrated biological samples.

It is shown that quantitative X-ray imaging of planed, frozen-hydrated, biological bulk samples that have not been etched is possible. X-ray imaging represents a better alternative to static beam (selected area) analysis of fractured frozen-hydrated samples. This procedure avoids the undesirable necessity of etching planed frozen-hydrated samples to provide an interpretable electron image. Qualitative oxygen and carbon X-ray images, which can be acquired in a short time, can be used for distinguishing morphological features and remove the requirement for electron images. In test samples of frozen-hydrated albumin, containing salts, analyses by X-ray images compared well with static beam (selected area) analyses from the same samples. An example of an analysis of frozen-hydrated insect Malpighian tubules is given in which the response to ouabain treatment was analysed. In this example X-ray imaging showed that ouabain resulted in a significant increase in cytoplasmic and luminal Na and a significant decrease in cytoplasmic and luminal K. X-ray imaging also showed that there was a significant increase in cellular water content. The presence of a potassium gradient in soybean root nodules was also demonstrated. The use of standard deviation images for processing low count images increases analytical precision but results in underestimates of the true concentrations.

Animals↗
PubMed1983

Chromatographic analysis of glutamic acid decarboxylase in biological samples.

A number of non-chromatographic and chromatographic methods for analysis of GAD activity in biological tissues have been described. The majority of these chromatographic methods utilize the analysis of GABA formed from incubation homogenates. Depending upon the analytical technique selected, limits of detection range from nanogram to picogram levels of GABA. Also discussed have been some of the commonly associated problems and their resolution with sample collection, postmortem changes and alternative pathways of CO2 and GABA production which can lead to errors in accurate determination of GAD activity in biological samples.

4-Aminobutyrate Transaminase↗