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PubMed1996

Endogenous alkaloids in man. XXVI. Determination of the dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) in biological samples using gas chromatography with selected ion monitoring.

Highly chlorinated beta-carbolines have a potential in vivo relevance to Parkinson's disease. In this paper, a gas chromatographic method for the determination of the neurotoxic 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), the condensation product of tryptamine and chloral hydrate, is described. The specific and sensitive assay involves purification of the biological samples by solid-phase extraction with C18 cartridges, derivatization with heptafluorobutyric anhydride, and chromatography on a non-polar fused-silica capillary column. Detection of TaClo was achieved by the registration of characteristic mass fragments of the TaClo heptafluorobutyric amide derivative using selected ion monitoring. The method was utilized to detect and quantify TaClo in blood, urine, bile, faeces, and brain tissue of rats treated with this alkaloid-type heterocycle. Four-fold deuterium-labelled TaClo was used as an internal standard.

Animals↗
PubMedDate not supplied

[Unified gas chromatography method for the detection of volatile toxicants in biological samples].

A method was designed to identify and quantify the volatile toxicants in cadaveric samples; it is based on analyzing the equilibrium of the gas-vapor phase. A system comprising 3 gas chromatography columns with different polarities and without preliminary isolation of an analyzed substance is used in toxicants' identification. 0.2% n-propanol solution was used in quantification of fresh biological samples, while methanol solution was used in cases of purulence-affected materials. The method sensitivity provides for detecting some of the toxicants, like acetaldehydes, acetone as well as ethyl and higher alcohols at the level of their natural content. The method has been tested for as long as 15 years at expertise objects.

Chromatography, Gas↗
PubMed1991

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.

Chromatography, High Pressure Liquid↗
PubMed1992

Glassy carbon pre-column for direct determination of acetylcholine and choline in biological samples using liquid chromatography with electrochemical detection.

The determination of acetylcholine and choline has been quite successfully accomplished using liquid chromatography with electrochemical detection following the original reports of Potter et al. [J. Neurochem., 41 (1984) 188]. A post-column reactor containing acetylcholinesterase and choline oxidase allows conversion of the desired species into hydrogen peroxide, an electrochemically active substance. However, the direct injection of tissue homogenates and other biological samples into such a system exhibits quite large solvent fronts and unidentified peaks. Using a pre-column packed with glassy carbon particles, we were able to dramatically decrease the size of the solvent front for such injections and tentatively identify the unknown peaks to be caused, at least in part, by common catecholamines. The glassy carbon pre-column, in addition to increasing the selectivity of the results, allowed the required chromatographic time per sample to be decreased from 20 to 10 min.

Acetylcholine↗
PubMed1997

Determination of dimethylamine in biological samples by high-performance liquid chromatography.

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 micromol/l. Recoveries from spiked serum samples were 99-107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3+/-1.5 and 29.1+/-12.1 micromol/l, respectively.

Adult↗
PubMed2002

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry.

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.

Animals↗
PubMed1988

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples.

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130 degrees C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6-40 mumol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3 +/- 2.9 and 89.4 +/- 2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.

Aldehyde Oxidoreductases↗
PubMed1985

Determination of 5-aminolevulinic acid in biological samples by high-performance liquid chromatography.

5-Aminolevulinic acid (ALA), the common precursor of all naturally occurring tetrapyrroles, forms a stable condensation product with 2-amino-3-hydroxynaphthalene which can be identified by its fluorescence. Separation of the compound by reversed-phase high-performance liquid chromatography on RPC-18 columns allows its detection down to the picomolar range and can be successfully applied for ALA analysis in small biological samples. The reaction product of ALA with 2-amino-3-hydroxynaphthalene has been synthesized and characterized.

Aminolevulinic Acid↗
PubMed2006

Analysis of zinc in biological samples by flame atomic absorption spectrometry: use of addition calibration technique.

The quantification of target analytes in complex matrices requires special calibration approaches to compensate for additional capacity or activity in the matrix samples. The standard addition is one of the most important calibration procedures for quantification of analytes in such matrices. However, this technique requires a great number of reagents and material, and it consumes a considerable amount of time throughout the analysis. In this work, a new calibration procedure to analyze biological samples is proposed. The proposed calibration, called the addition calibration technique, was used for the determination of zinc (Zn) in blood serum and erythrocyte samples. The results obtained were compared with those obtained using conventional calibration techniques (standard addition and standard calibration). The proposed addition calibration was validated by recovery tests using blood samples spiked with Zn. The range of recovery for blood serum and erythrocyte samples were 90-132% and 76-112%, respectively. Statistical studies among results obtained by the addition technique and conventional techniques, using a paired two-tailed Student's t-test and linear regression, demonstrated good agreement among them.

Calibration↗
PubMed2006

TopCount coupled to ultra-performance liquid chromatography for the profiling of radiolabeled drug metabolites in complex biological samples.

The recent commercial availability of small particle packed columns (<2microm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography (UPLC). It has recently been shown that the improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of TopCount, a microplate scintillation counter, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is tested. TopCount, has innumerable benefits over more traditional on-line radioactivity flow detection methods, when dealing with the narrow peak widths and small peak volumes associated with the enhanced efficiency of sub-2microm columns. The system is tested for robustness and sensitivity, and then used to undertake successful metabolite profiling of actual samples, and the data compared to traditional HPLC with on-line radioactivity flow detector.

Animals↗
PubMed1997

Determination of organophosphorous pesticides in biological samples of acute poisoning by HPLC with diode-array detector.

We have developed a simple and rapid method for measuring 11 organophosphorous pesticides (dichlorvos, methidathion, salithion, malathion, fenitrothion, fenthion, parathion, diazinon, ethylthiometon, O-ethyl O-(4-nitrophenyl)phenylphosphonothioate (EPN) and chlorpyrifos) and one metabolite (3-methyl-4-nitrophenol) of fenitrothion in serum and urine of acute poisoning patients by HPLC with a diode-array detector. An aliquot of the biological sample after deproteinization by acetonitrile was injected into C18 column using acetonitrile-water as a mobile phase. The detection limits in serum and urine ranged from 0.05 to 6.8 micrograms/ml at a wavelength of 230 nm. This method was successfully applied to two actual cases of acute poisoning.

Aged↗
PubMed2006

Lead ultra-trace on-line preconcentration and determination using selective solid phase extraction and electrothermal atomic absorption spectrometry: applications in seawaters and biological samples.

In this work, a new chelating resin [1,5-bis (2-pyridyl)-3-sulphophenyl methylene] thiocarbonohydrazide immobilised on aminopropyl-controlled pore glass (550 A; PSTH-cpg) was synthesised and packed in a microcolumn which replaced the sample tip of the autosampler arm. The system was applied to the preconcentration of lead. When microliters of 10% HNO3, which acts as elution agent, pass through the microcolumn, the preconcentrated Pb(II) is eluted and directly deposited in a tungsten-rhodium coated graphite tube. With the use of the separation and preconcentration step and the permanent modifiers, the analytical characteristics of the technique were improved. The proposed method has a linear calibration range from 0.012 to 10 ng ml(-1) of lead. At a sample frequency of 36 h(-1) with a 90 s preconcentration time, the enrichment factor was 20.5, the detection and determination limits were 0.012 and 0.14 ng ml(-1), respectively and the precision, expressed as relative standard deviation, was 3.2% (at 1 ng ml(-1)). Results from the determination of Pb in biological certified reference materials were in agreement with the certified values. Seawaters and other biological samples were analysed too.

Calibration↗
PubMedDate not supplied

RT-nested PCR for the detection of enterovirus in biological samples from patients with suspected enteroviral infections.

In this study, we have tested a reverse transcription (RT) nested polymerase chain reaction (nPCR) for detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF), serum samples, and conjunctival swabs (CS) from patients with suspected enterovirus infections. A specific 113-bp fragment was amplified using primers designed based on 5' non coding region of the enterovirus genome. The enterovirus RT-nPCR was able to detect 0.001 plaque forming unit (pfu)/ml. Since no PCR product was detected in each of the CSF, CS and serum samples from patients with proven-non-enterovirus viral infections, this method was found to be specific. EV RNA was detected in all 30 culture-confirmed CSF samples and yielded positive results in 5 out of 7 additional cases of culture-negative CSF samples with other evidences of enterovirus infection. Overall, EV RNA was detected in 95% of the patients with clinical diagnosis of viral central nervous system (CNS) disease and confirmed enterovirus infection. Furthermore, we were able to detect EV RNA in 24 (47%) out of 51 CSF samples from patients with clinical diagnosis of viral CNS disease and negative laboratory evidence of viral infection. The percentage of positive EV RNA detection in paired CSF and serum samples from 11 patients with an enterovirus isolate in CSF was 100% (11 of 11) and 73% (8 of 11), respectively. In addition, EV-specific IgM was detected in 64% (7 of 11) of the sera tested. The method was also tested against 136 samples of CS from patients with clinical diagnosis of acute hemorrhagic conjunctivitis. Ninety nine of them resulted positive (73%), while only 27 (20%) had been positive for viral culture. In summary, our study shows the importance of enterovirus RT-nPCR for the diagnosis of enterovirus associated disease in different kind of biological samples and different types of diseases.

Acute Disease↗
PubMed2006

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection.

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.

Animals↗
PubMed2006

Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples.

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.

Antioxidants↗
PubMed2004

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples.

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.

Gas Chromatography-Mass Spectrometry↗
PubMed2006

Nanometer-scale heat-conductivity measurements on biological samples.

With semiconductor structures reaching the nanometer scale, heat conductivity measurements on the mesoscopic range of some tens of nanometers become an increasingly important aspect for the further improvement in digital processing and storage. Also the attempt to use atomic-force microscopy (AFM) technology for high-density data storage by writing information bits as nanometer-sized indentations into a polymer substrate with a heated cantilever tip asks for a careful investigation of the nano-scale heat-conductivity properties of polymers. Furthermore, in many AFM imaging applications, heat conductivity can provide additional information about the material the imaged structures consist of. In this respect, heat conductivity can also become very interesting in studies of usually quite heterogeneous biological samples, if the resolution can attain the nanometer range. In standard scanning thermal microscopy application, the tip forms a thermocouple, which precludes high-resolution imaging, as thermocouples cannot be made sufficiently small. In this paper, which focuses on biological applications, we demonstrate that by using an ultra sharp AFM cantilever with a Joule heating element above the tip structure different molecular components can be distinguished thanks to their different heat-conductivity properties. In this case, the resolution is determined by the actual tip size, and it can reach 10nm.

Collagen↗
PubMed1997

Quantitative gas chromatography-mass spectrometry isomer-specific measurement of hydroxy fatty acids in biological samples and food as a marker of lipid peroxidation.

We have developed a capillary gas chromatography-mass spectrometry method for the quantitative analysis of individual positional isomers of monohydroxy fatty acids derived from linoleic, arachidonic, eicosapentaenoic, or docosahexaenoic acid. Peroxidation of a particular polyunsaturated fatty acid results already in a complex mixture of positional isomers of hydroperoxy and hydroxy fatty acids. Catalytic hydrogenation of lipid extracts produces stable saturated hydroxy lipids from the complex mixtures typical of oxidized biological samples, simultaneously simplifying the analytical problem and eliminating oxidation artifacts. After saponification and methylation, monohydroxy fatty acid methyl esters are purified by solid-phase extraction and partially resolved using a CP Sil 19 column following on-column derivatization of the hydroxy groups with tetramethylammonium hydroxide. The resulting methoxy fatty acid methyl esters are subjected to electron impact mass spectroscopy. Two characteristic ions are produced for each positional isomer. Quantitative measurements were achieved by using odd chain C17 and C19 monohydroxy fatty acids as internal standards. The limit of detection of individual hydroxy fatty acid isomers is dependent on the total number of ions monitored. Monitoring 11 pairs of ions simultaneously gives limits of detection of 10 ng. Sensitivity is much higher by monitoring fewer ions and as little as 0.2 ng of a single isomer can be detected. The method has been applied for the quantitative analysis of hydroxy (plus hydroperoxy) fatty acids in plasma, adipose tissue, oils, and foods. To date over 1000 samples have been analyzed using the method described in this paper.

Dietary Fats, Unsaturated↗