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PubMed2004

Mass spectrometric methods for the determination of flavonoids in biological samples.

There is an ever-increasing interest in the biological effects of the bioflavonoids, members of the large group of plant polyphenols. Because of the aromatic character of these compounds, they have been analyzed by several chromatographic methods. In the case of high-performance liquid chromatography, they are readily detected by their ultraviolet absorbance or electrochemical properties. More evidence that the bioflavonoids undergo extensive metabolism during uptake from the gut and distribution around the body and in specific tissues is accumulating. In addition, free radical products at sites of inflammatory processes react with bioflavonoids and their metabolites, generating important new compounds of as yet unknown properties. For these reasons, careful examination of the chemical nature of bioflavonoids and their products in biological systems is absolutely required. Combination of mass spectrometry with the various chromatographic methods has proved to be highly successful in this regard. This review of the literature on the bioflavonoids is focused on the methods that are currently available for their qualitative and quantitative analysis by mass spectrometry and covers the period 2001-2003. Emphasis is placed on the description and value of existing methods, followed by an examination of emerging technologies.

Animals↗
PubMed2002

An enzymatic method of analysis for GDP-L-fucose in biological samples, involving high-performance liquid chromatography.

To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.

Aminopyridines↗
PubMed1981

Techniques for the measurement of methotrexate in biological samples.

Techniques available for the measurement of methotrexate (MTX) in biological fluids are reviewed. The importance of measuring MTX in plasma following high-dose MTX treatment is now well recognized and renewed interest in the metabolism and pharmacokinetics of the drug has prompted the development of new techniques of analysis. Results obtained with our radioimmunoassay are presented and compared with results obtained by other methods.

Animals↗
PubMed1975

A micro method for the determination of volatile metabolites in biological samples. A preliminary report.

A new method for analyzing volatile metabolites in biological fluids and tissues has been developed. The procedure involves extraction of the sample with diethyl ether, concentration on a short column of glass wool by elution of the diethyl ether, then thermal desorption of the trace constituents into a chromatographic system. Small samples of 100 mul are quite adequate. The method is applicable to serum, plasma, urine and other fluids. Small tissue samples can also be used with this technique.

Animals↗
PubMed1985

Simultaneous determination of histamine and N alpha-methylhistamine in biological samples by an improved enzymatic single isotope assay.

A modified highly sensitive and specific radioenzymatic assay for the simultaneous determination of histamine (HA) and N alpha-methylhistamine (N alpha-MH) in tissues and body fluids is described. In the presence of the enzyme histamine-N-methyltransferase and of the methyl donor [3H]-S-adenosylmethionine, the transmethylation process was about seven times more effective for HA than for N alpha-MH. In the same conditions only very low amounts of N alpha, N alpha-dimethylhistamine (N alpha, N alpha-DMH) were converted into its 1-methylated derivative. The high degree of specificity attained by this method is due to the rapid quantitative extraction of the biological fluids, to the partially purified enzyme preparation and to the thin-layer chromatography system used which allows an excellent separation of the 3H-1-methyl products of HA, N alpha-MH and N alpha, N alpha-DMH. This method is highly sensitive for the assay of HA and N alpha-MH (detection limit 10 and 50 picograms, respectively), but due to lack in sensitivity, it cannot be extended to the measurement of N alpha, N alpha-DMH. The HA content of 20-30 samples can be determined in duplicate by one person in a working day. The concentrations of HA measured by this method in different biological samples (human whole blood, plasma, urine, gastric juice and skin biopsies) were in good agreement with the values reported in the literature. The presence of minute amounts of N alpha-MH in the human gastric juice was established by rigorous checking.

Body Fluids↗
PubMedDate not supplied

Lipid extraction from blood and biological samples and concentrations of dioxin-like compounds.

The extraction of lipid from human blood and other biological materials in the determination of dioxin-like and organochlorine compounds has been studies using different amounts of extracting solvent. When large volumes of ethanol-hexane are used to extract small blood samples, the total lipid extracted is incomplete as the polar phospholipids do not partition into the organic phase. This failure to extract all the blood lipid results in an inaccurately high concentration expressed on a lipid basis and, by corollary, a high biased body burden. This solvent dependence of lipid extraction does not appear to be a problem for most other biological and food samples since over 90% of the lipid is in the form of readily extractable neutral glycerides.

Adipose Tissue↗
PubMed1993

The use of cyclic voltammetry for the evaluation of oxidative damage in biological samples.

A method using cyclic voltammetry to evaluate oxidative damage in biological systems is presented. Three biological systems were tested: Escherichia coli cells, the rat jejunal mucosa, and the enzyme, lactate dehydrogenase. Exposure of E. coli cells to oxidative stress resulted in a rapid decrease in their survival and a decrease in their ability to accumulate 14C-leucine. This was accompanied by a significant increase in the oxidation potential of the cells. Similar results were obtained when the rat jejunal mucosa was exposed in a perfusion system to oxidative stress induced by the hydroxyl radical produced by either hydrogen peroxide and ferrous ions or the combination of ascorbic acid and copper ions. Loss of cellular potassium was taken as an indication of damage to the rat jejunum. Exposure of lactate dehydrogenase to oxidative stress induced by hydroxyl and peroxyl radicals also resulted in a significant loss of enzyme activity along with a pronounced change in the cyclic voltammogram of the enzyme. It was concluded that measurement of the oxidation potentials of these biological systems can give an indication of the occurrence of oxidative damage.

Animals↗
PubMed1998

Methods for the measurement of benzodiazepines in biological samples.

A review of methods for the measurement of benzodiazepines in biological specimens published over the last five years is presented. A range of immunoassay procedures using EIA, ELISA, FPIA, agglutination or kinetic interaction of microparticles, or RIA methods are now available. Cross reactivities to benzodiazepines are variable such that no one kit will recognise all benzodiazepines and their relevant metabolites at concentrations likely to be encountered during therapeutic use. Prior hydrolysis of urine to convert glucuronide metabolites to immunoreactive substances improves detection limits for many benzodiazepines. Several radioreceptor assays have now been published and show good sensitivity and specificity to benzodiazepines and offer the advantage (over immunoassay) of being able to detect these drugs with equal sensitivity. Solvent extraction techniques using a variety of solvents were still popular and offer acceptable recoveries and lack of significant interference from other substances. A number of papers describing solid phase extraction procedures were also published. Direct injection of specimens into a HPLC column with back flushing were also successfully described. Seventy two chromatographic methods using HPLC, LC-MS, GC and GC-MS methods were reviewed. HPLC was able to achieve detection limits for many benzodiazepines using UV or DAD detection down to 1-2 ng/ml using 1-2 ml of urine or serum (blood). ECD detectors gave detection limits better than 1 ng/ml from 1 ml of specimen, which was an order of magnitude lower than for NPD. EI-MS offered similar sensitivity, whilst NCI-MS was capable of detection down to 0.1 ng/ml. Methods suitable for the separation of enantiomers of benzodiazepines have been described using HPLC. Electrokinetic micellar chromatography has also been shown to be capable of the analysis of benzodiazepines in urine.

Benzodiazepines↗
PubMed1993

Sample cleanup by solid-phase extraction for the ultratrace determination of polychlorinated dibenzo-p-dioxins and dibenzofurans in biological samples.

A sample cleanup method for the isolation of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) by solid-phase extraction is described. A C18 bonded silica cartridge is used for the initial extraction and enrichment of the analytes. Subsequent cleanup is achieved with a dual-cartridge arrangement consisting of a bonded benzenesulfonic acid cartridge in series with a silica cartridge. A Florisil cartridge is employed for the final cleanup step. Sample preparation procedures used for blood plasma and animal tissues are described. The precision and accuracy of the method is validated by determining recoveries of 2,3,7,8-substituted PCDD and PCDF congeners spiked at various levels in blood plasma and in animal tissues. The effectiveness of the cleanup method is shown by the analysis of wild bird eggs which are highly contaminated with a variety of pesticides and polychlorinated biphenyls. The application of this cleanup method to human plasma containing PCDDs/PCDFs at parts-per-quadrillion levels and results of analyses by high-resolution mass spectrometry and low-resolution mass spectrometry are presented. The validity of this method is demonstrated by the comparable results obtained from this method and from other established methods.

Animals↗
PubMed1989

[Critical aspects in determining total radioactivity of biological samples].

During measurements of radioactivity in some milk samples with liquid scintillation counter (about one year after the nuclear accident of Chernobyl) we have observed an increase of the values of scintillation fluid with the passing of time. Although this enhancement is absolutely small (about 2 c.p.m. in 500 min), it is very important for an exact measurement of samples at low counting, as those tested. Our protocol of measure provides for insertion of alternate blanks and samples in the automatic sample-holders of liquid scintillation counter. The values of measurement of samples are taken during the increase phase subtracting the value of blank interpolated on the increasing straight line from c.p.m. of sample. Finally, we report the collected values of the whole radioactivity in some milk samples: at least 5-6 nCi/L contrary to about 1 nCi/L of 137Cs reported by USL. In our opinion it is important to consider the whole radioactivity as measure of the overall biological danger of radioactive samples. In fact, this measurement takes into account also biologically very dangerous radionuclides as 3H, 14C, 90Sr.

Animals↗
PubMed1990

Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples.

Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.

Acetylene↗
PubMed1991

Rapid and sensitive high-performance liquid chromatographic method for the analysis of tryptophan, tyrosine and phenylalanine in biological samples.

In order to overcome problems related to the determination of free tryptophan in biological fluids using conventional methods, we have developed an accurate and reliable procedure based on a specific pretreatment of samples followed by a very rapid and sensitive reversed-phase high-performance liquid chromatographic analysis. The pretreatment consists of adding to the samples of a very low amount of 3 M phosphate buffer to maintain pH in the physiological range followed by ultrafiltration. The precision, reproducibility and sensitivity of our method were also evaluated. The recovery of each amino acid was greater than 92%. The use of a microbore column allows the detection of up to 0.2 pmol/microliter of amino acid. The method has been applied to the analysis of samples obtained from 25 normal and 10 phenylketonuric subjects.

Chromatography, High Pressure Liquid↗
PubMed2000

Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples.

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.

Autopsy↗
PubMed1997

Ozone degradation of residual carbon in biological samples using microwave irradiation.

In an attempt to produce complete oxidation of a biological matrix, bovine liver, ozone was investigated as an additional, potentially non-contaminating, oxidizing reagent after nitric acid digestion. Experiments were carried out to determine the decomposition efficiency of residual carbon species, primarily o-, m- and p-nitrobenzoic acids (NBAs), using ozone. The NBAs were degraded by purging sample digests with ozone, while heating the solutions with microwave energy at atmospheric pressure. The effects of the degradation temperature and solution pH on the ozonation of NBAs were determined. Solid phase extraction (C18) was used to extract NBAs from the acid digestate solutions prior to HPLC analysis. Reversed phase HPLC was used to determine NBA concentrations in digest solutions. After 2.5 h of purging ozone at 80 degrees C, 33.65 +/- 3.80% o-NBA degradation, 19.39 +/- 1.74% m-NBA degradation, and 26.47 +/- 3.36% p-NBA degradation were obtained.

Animals↗
PubMed1988

Measurement of copper in biological samples by flame or electrothermal atomic absorption spectrometry.

Guidelines presented here allow for copper analysis of biological materials by methods that are very sensitive, that require little sample preparation, that have few chemical or spectral interferences, that are inexpensive, and that require only usual care in contamination control. The commercial instruments for FAAS and ETAAS from Perkin-Elmer, from Varian, and from Instrumentation Laboratories Inc. (Allied Analytical Systems) all work well in either the flame or the flameless mode. Background correction techniques are not essential for copper analysis if care is taken with the sample preparation to minimize the background signals. Different types of burners will work adequately if one makes certain that the viscosity of the sample and the control products are similar to the calibration standards. Further, dilution of samples is preferred over increasing the viscosity of the calibration standards by the addition of a protein containing solution or a substance such as glycerol. A 1:10 dilution of blood plasma or serum with dilute nitric acid or water is all that is necessary for copper analysis by the FFAS methods. Cation and anion effects should be tested by bracketing the concentrations of the ions found in the sample with known amounts of ions in the sample solutions. Increasing the concentrations of the ions thought to interfere while keeping the copper concentration constant is another way to test for ion interferences.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗