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PubMed2002

Rapid quantitation of globotriaosylceramide in human plasma and urine: a potential application for monitoring enzyme replacement therapy in Anderson-Fabry disease.

A method for measuring globotriaosylceramide (Gb3, or GL3) levels in plasma and urine of humans affected by Anderson-Fabry disease has been developed. The analyses are performed using flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS). The method is rapid, sensitive and hence suitable for high-throughput analyses, requiring only a simple 50-fold dilution for the preparation of plasma and urine samples. The detection of the analytes of interest was achieved using a triple quadrupole instrument operating in the multiple reaction monitoring mode. The linearity of the calibration standard responses, the intra- and inter-assay precision, the accuracy and the detection limit of the method were evaluated. The proposed method allows a rapid and accurate assessment of globotriaosylceramide in biological samples. Data obtained from healthy volunteers and Anderson-Fabry affected subjects suggest a potential role for this technique in monitoring the effectiveness of Anderson-Fabry disease therapy. The results obtained in two actual cases treated with enzyme replacement therapy are reported and discussed.

Fabry Disease↗
PubMed1999

Chemical characterization and screening of hydrocarbon pollution in industrial soils by headspace solid-phase microextraction.

A headspace solid-phase microextraction method, followed by a gas chromatographic-mass spectrometric analysis, has been developed for the screening of soil samples polluted by coal tar or refined petroleum products. Vapor pressures of target analytes were determined using a capillary GC method to identify environmentally important components with a sufficiently high vapor pressure to be analyzed in the headspace mode. The method was optimized under non-equilibrium conditions with simplicity and automation in mind and does not require any extraction procedure or sample preparation, other than grinding, drying and homogenizing. The analytical performance and the significance of the results for the purpose of chemical characterization, source discrimination, determination of individual isomer distributions and to calculate source or weathering ratios, is discussed.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

An activated carbon substrate surface for laser desorption mass spectrometry

A method to obtain laser desorption/ionization mass spectra of organic compounds by depositing sample solutions onto a carbon substrate surface is demonstrated. The substrate consists of a thin layer of activated carbon particles immobilized on an aluminum support. In common with the porous carbon suspension samples used in previous "surface-assisted laser desorption/ionization" (SALDI) work, the mass spectra contain only a few "matrix" background ion peaks, minimizing interference with analyte ion peaks. The presence of glycerol ensured that the ion signals were stable over hundreds of laser shots. In addition, the carbon substrate surface has several advantages over the suspension samples. The use of a very thin layer of carbon significantly improves the sensitivity. Detection limits range from attomoles for crystal violet to femtomoles for bradykinin. Very little sample preparation is required as the analyte solution is simply pipetted onto the substrate surface and glycerol added. When using an alternate sample deposition method, a mass resolution for bradykinin of 1800 is achieved in linear time-of-flight mode. This is close to the resolution limit set by the detector system and above instrument specification for matrix-assisted laser desorption/ionization mass spectra.

Journal Article↗
PubMed1984

Laser microprobe mass spectrometry of platinum in dog kidney after cisplatin administration.

By means of laser microprobe mass analysis ( LAMMA ) platinum was detected in the renal proximal tubular cells of a dog that had been intravenously administered the antitumor drug cisplatin (5 mg per kg body weight). No definite subcellular localization of the heavy metal was obtained. Sample preparation and analytical features are examined to increase spatial resolution of analysis while maintaining sufficient detection efficiency. The LAMMA method is destructive, but the amount and type of evaporated material can readily be determined when using LAMMA in combination with transmission electron microscopy. Instrumental optimization and standardization of mass signals is possible by using platinum-loaded, ion-chelating resin beads embedded and sectioned with the tissue.

Animals↗
PubMed1995

Determination of intracellular levels of 6-mercaptopurine metabolites in erythrocytes utilizing capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH2PO4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (lambda ex = 325 nm, lambda em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 microliters red blood cells for the three analytes (RSDs < or = 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

Electrophoresis↗
PubMed1990

Simultaneous multielement determination in vegetable foodstuffs and their respective cell fractions by total-reflection X-ray fluorescence (TXRF).

Total-reflection X-ray fluorescence (TXRF) was employed in the multielement determination made on samples of lamb's lettuce and cauliflower as well as in the analysis of their soluble and insoluble cell fractions. All samples were digested with HNO3 and the elements were quantitatively determined with Ga as internal standard. For cell fractionation, the freeze-dried vegetables were mortared in the presence of fine-grain quartz and extracted with a buffer solution; the resulting suspension was then separated by ultracentrifugation into cytosol and pellet components. K, Ca, Mn, Fe, Cu, Zn, Rb and Sr were the elements of which the total content and distribution between cytosol and pellet were determined. As a result of the cellular digestion and extraction procedures employed, greater than or equal to 50% of the total metal contents of Zn, Cu, K and Rb could be reduced to the cytosol phase in both vegetables, however, Sr, Fe, Ca and Mn were mainly bound to the insoluble pellet components which, in the case of cauliflower, contained up to 100% of the total Sr content. As a multielement method, TXRF proved to be an excellent analytical tool in these investigations, since it requires only minute samples with simple preparation and involves a large dynamic measuring scale.

Calcium↗
PubMedDate not supplied

Applicability of microwave acid digestion to sample preparation of biological materials for analysis by particle-induced X-ray emission (PIXE).

A microwave acid digestion method for the preparation of biological samples for PIXE analysis is presented. The precision and accuracy of the entire PIXE analytical procedure, including the microwave digestion step, were evaluated by analyzing eight certified reference materials. For elements heavier than K, and for concentration levels from 2 micrograms/g upward, the total random error of a single analysis is in the range of 2-5%. The accuracy is better than 5%. The detection limits are down to 0.3 micrograms/g.

Acids↗
PubMed2000

Comparison of the levels of 8-hydroxyguanine in DNA as measured by gas chromatography mass spectrometry following hydrolysis of DNA by Escherichia coli Fpg protein or formic acid.

8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possibility of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua by GC/MS. Derivatization has been reported to produce artifacts by oxidation of guanine to 8-OH-Gua in acid-hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivatization of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization of acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results with the results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented.

Animals↗
PubMed2005

Gas chromatography/ion trap mass spectrometry applied for the determination of polybrominated diphenyl ethers in soil.

A gas chromatography/ion trap mass spectrometry (GC/ITMS) method was developed for the determination of polybrominated diphenyl ethers (PBDEs). ITMS parameters were optimized in order to achieve the best sensitivity for the PBDE analysis. Tandem mass spectrometry, along with an isotope dilution internal standard method, was used for the quantitation. Chromatographic windows were developed for mono- to hepta-BDEs, depending on the retention times when a 30-m GC column was used. A different 15-m column was used to analyze deca-BDE. Environmental soil samples collected from an electronic waste recycling site were prepared by using Soxhlet extraction and column chromatographic cleanup. Average recoveries of 61-118% were obtained for the 13C-labeled PBDE internal standards spiked in the samples prior to sample preparation. The accuracy represented by relative analytical errors was -24% to 18%, and the precision (relative standard deviation) was 11-26% (n=8). The method detection limits ranged from 0.013-0.25 ng/g for the PBDEs in soil.

Gas Chromatography-Mass Spectrometry↗
PubMed1981

Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels.

The ribonucleases (RNases) of human blood serum, urine, cerebrospinal fluid (CSF), and leukocytes were visualized by activity staining after electrophoresis in RNA-case sodium dodecyl sulfate-polyacrylamide gels. Samples were prepared for electrophoresis by heating for 2 min at 100 degrees C in 2% sodium dodecyl sulfate (NaDodSO4) and 5% mercaptoethanol, conditions which dissociate proteins into their constituent polypeptide chains and permit estimation of molecular weight. It was found that each of the five peaks of serum alkaline RNase activity separable on phosphocellulose columns, i.e., RNases 1-5 of Akagi et al. [Akagi, K., Murai, K., Hirao, N., & Yamanaka, M. (1976) Biochim. Biophys. Acta 442, 368-378], is associated with electrophoretically distinct enzymes. The molecular weights exhibited by these enzymes in NaDodSO4 gels are 31 000 and 28 000 (major species of RNase 1), 25 000 (RNase 2), 20 000 (RNase 3), 16 000 (RNase 4), and 14 000 (RNase 5). The RNase activity of leukocytes displays a molecular weight of 17 000 and exhibits a characteristic dependence of its Rf on the temperature at which samples (in 2% NaDodSO4 without mercaptoethanol) are prepared for electrophoresis. An RNase activity like that of leukocytes, distinct from RNases 1-5, is found in serum. Urine RNase activity is less heterogeneous than that of serum, consisting mainly of species like serum RNase 1 and an enzyme similar to leukocyte RNase. Conversely, CSF RNase activity is more complex and includes enzymes resembling serum RNases 1-5 as well as additional species either not observed in serum or detected in serum as minor components following chromatography. The analytical methods described herein are particularly useful for assessment of heterogeneity of RNase preparations and for direct comparison of the RNases of crude and purified samples.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Sensitive determination of the phosphodiesterase III/IV inhibitor zardaverine in human serum by direct sample injection, automated precolumn clean-up and high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of the phosphodiesterase III/IV inhibitor zardaverine in serum by using fully automated clean-up of large-volume serum samples on a semi-preparative-scale precolumn followed by chromatography on two analytical columns operated with two different solvent systems. The switching of the analytical columns provides the necessary specificity and sufficient sensitivity for UV detection is obtained by the sample volume. The method was shown to give nearly quantitative recovery, allowing the use of external standard quantification. Good precision and linearity within the concentration range 1-50 ng/ml could be demonstrated. The method is suitable for routine measurements in support of kinetic studies of zardaverine in man.

Chromatography, High Pressure Liquid↗
PubMed2005

Multi-residue determination of anti-inflammatory analgesics in sera by liquid chromatography--mass spectrometry.

Non-steroidal anti-inflammatories (NSAIDs) are analgesic, antipyretic, and, as their name implies, anti-inflammatory drugs, which are widely used for the treatment of a variety of human and veterinary disease conditions in which control of pain and inflammation is desired. Acetaminophen (ACE) is a common over-the-counter analgesic. Detection of a variety of widely used NSAIDs and ACE in fluid and tissue samples is an important diagnostic tool. A sensitive and selective analytical method has been developed for simultaneous screening of 12 NSAIDs and ACE by liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface set to operate in the negative ion mode of MS. Following sample preparation, all analytes were separated on a C18-reversed-phase column with a gradient elution of acetonitrile and acetic acid. Full-scan mass spectral fragmentation profiles were established for each analyte and individual extracted ion chromatograms were used for quantitation. Linearity of detection was observed over the 0.05-25.0 microg/mL range of standard concentrations. The instrument limits of detection (LOD), based on an individual analyte quantitation ions, fell between 0.05 and 1.0 microg/mL for all compounds. The matrix LODs were determined to be 0.05 microg/mL for phenylbutazone (m/z 307); 0.1 microg/mL for indomethacin (m/z 312), flunixin (m/z 295), and piroxicam (m/z 330); 0.5 microg/mL for ACE (m/z 150), diclofenac (m/z 250), ketoprofen (m/z 209), and mefenamic acid (m/z 240); 1.0 microg/mL for oxyphenbutazone (m/z 323); 5.0 microg/mL for ibuprofen (m/z 205), salicylic acid (m/z 137), and tolmetin (m/z 212); and 10 microg/mL for naproxen (m/z 185).

Acetaminophen↗
PubMed2003

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching liquid chromatography with electrospray tandem mass spectrometry.

Using column-switching liquid chromatography/tandem mass spectrometry (LC-MS/MS), we developed an improved analytical method of urinary estriol glucuronides. This new method is derived predominantly from maternal and fetal precursors in pregnancy. We used in the following procedure: first, we filtered urine samples with a membrane filter. Next, we directly injected the 50 microL aliquot of urine samples onto a pre-column. Then, after activating the column-switching valve, we backflushed the loaded samples onto the C(18) analytical column. Urine samples can be assayed within 20 min without any sample preparation steps. We monitored separated estriol glucuronides by negative electrospray ionization (ESI) and selected-reaction monitoring (SRM). The calibration range of estriol-3-glucuronide (E3-3G) and estriol-16-glucuronide (E3-16G) was 0.1-20 microg/mL and the linearity of the method was 0.9984 for E3-3G and 0.9987 for E3-16G. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng/mL (E3-3G) and 5 ng/mL (E3-16G). The analytical recovery was over 85% and, in general, inter-day and intra-day variability for precision and accuracy were less than 10%. When applied to a pregnancy urine sample to biomedical monitoring of the function of the maternal/fetal unit, the proposed method allowed rapid and sensitive screening for the detection of E3-3G and E3-16G.

Adult↗
PubMed2006

On-line sample extraction and enrichment of non-steroidal anti-inflammatory drugs by pre-column in capillary liquid chromatography mass spectrometry.

A rapid and sensitive analytical method has been developed for the simultaneous determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma by capillary liquid chromatography (LC) and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The sample clean-up and enrichment on a pre-column were accomplished on-line to improve the sensitivity. This method greatly reduced sample preparation time and sample volume compared with off-line sample extraction methods and conventional LC methods, respectively. The recoveries of NSAIDs from human plasma were 56.7-96.9%. The total analytical time for a single analytical run was approximately 15 min. The detection limits of NSAIDs were 0.001-0.075 microg ml(-1) using a selected ion monitoring mode.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMedDate not supplied

Quantitation of the sulfur mustard metabolites 1,1'-sulfonylbis[2-(methylthio)ethane] and thiodiglycol in urine using isotope-dilution Gas chromatography-tandem mass spectrometry.

Sulfur mustard (HD), or bis(2-chloroethyl)sulfide, has several urinary metabolites that can be measured to assess human exposure. These metabolites include the simple hydrolysis product thiodiglycol (TDG) and its oxidative analogue, TDG-sulfoxide, as well as metabolites of the glutathione/b-lyase pathway 1,1'-sulfonylbis[2-(methyl-sulfinyl)ethane] (SBMSE) and 1-methyl-sulfinyl-2-[(methylthio)ethyl-sulfonyl]ethane (MSMTESE). Current methods focus on either the TDG or the b-lyase metabolites. We have developed a single method that measures products of both metabolic branches, with the reduced compound of SBMSE and MSMTESE, 1,1'-sulfonylbis [2(methylthio)ethane] (SBMTE), as the definitive analyte and TDG as a confirmation analyte. Sample preparation included b-glucuronidase hydrolysis for TDG-glucuronide conjugates, titanium trichloride reduction of sulfoxides to SBMTE and TDG, solid-phase extraction, and a chemical derivatization. We analyzed samples using gas chromatography-tandem mass spectrometry with quantitation using isotope-dilution calibration. The method limits of detection for TDG and SBMTE were 0.5 ng/mL and 0.25 ng/mL, respectively, with relative standard deviations of less than 10%. Urine samples from individuals with no known exposure to mustard agent HD had measurable concentrations of TDG, but no SBMTE was detected. The geometric mean concentration of TDG was 3.43 ng/mL, with concentrations ranging from < 0.5 ng/mL to 20 ng/mL.

Animals↗
PubMed1976

Facilitated purification of hypoxanthine phosphoribosyltransferase.

Three major approaches to the complete purification of hypoxanthine phosphoribosyltransferase from human erythrocytes and rat brain are described. Preparative isoelectric focusing which has been used for the isolation of the human enzyme was not fully successful in the case of rat brain. Preparative polyacrylamide-gel electrophoresis in gel blocks yields enzyme samples of high purity as judged by analytical gel electrophoresis, but with a comparatively low specific enzyme activity. The most rapid and convenient method, a modification of the affinity chromatography on GMP agarose first described by Hughes[5] gives hypoxanthine phosphoribosyltransferase which is superior to the other preparations in its homogeneity and its specific activity. All three methods produce an identical enzyme protein detected by polyacrylamide electrophoresis on nondenaturing and sodium dodecylsulfate gels. Molecular data of hypoxanthine phosphoribosyltransferase derived from these studies are: Isoelectric points of 5.60; 5.85 and 5.90 for three isozyme peaks of the rat brain enzyme; and a molecular weight of 72000 for the native rat brain enzyme and of 25000-27000 for the subunit of human and rat enzyme. Guanylate kinase does not interfere with the purification of hypoxanthine phosphoribosyltransferase on GMP agarose and moreover is itself partially purified by this chromatography.

Animals↗
PubMed2000

Evaluation of ursodeoxycholic acid bioavailability from immediate- and sustained-release preparations using gas chromatography-mass spectrometry and high-performance liquid chromatography.

An improved procedure is presented for the determination of ursodeoxycholic acid (CAS 128-13-2, UDCA) in human plasma and bile after oral administration of UDCA-containing dosage forms. The plasma samples after solid-phase extraction with silica-based C18- and strong anion exchange cartridges were assayed by gas chromatography-mass spectrometry (GC-MS) using selected-ion monitoring. The hexafluoroisopropyl trifluoroacetate ester derivative of UDCA was selected for GC analysis since it is easily and rapidly prepared by a one-step reaction. Biliary UDCA levels were determined by a rapid and simple high-performance liquid chromatographic (HPLC) method with on-line sample purification. This analytical protocol was used to investigate the pharmacokinetic of a new sustained-release capsule of UDCA in comparison with a reference immediate-release preparation after single oral administration. Statistical evaluation of the area under the plasma concentration-time curves indicated that two formulations are equivalent with regard to the amount of drug absorbed. However, pharmacokinetic data showed that with the sustained-release preparation a significantly delayed mean peak plasma level was reached compared with the reference preparation. Moreover, the immediate- and extended-release capsules were found to achieve a comparable degree of biliary enrichment with UDCA.

Adult↗
PubMed2000

Laboratory database to manage electrophoresis and chromatography separations and the associated samples.

A database was developed to store, organize, and retrieve the data associated with electrophoresis and chromatography separations. It allows laboratories to store extensive data on separation techniques (analytical and preparative). The data for gel electrophoresis includes gel composition, staining methods, electric fields, analysis, and samples loaded. The database stores data on chromatography conditions, the samples used, and the fractions collected. The data structure of this database was designed to maintain the link between samples (including fractions) from chromatography separations and their analysis by gel electrophoresis. The database will allow laboratories to organize and maintain a large amount of separation and sample data in a uniform data environment. It will facilitate the retrieval of the separation history of important samples and the separation conditions used.

Chromatography↗