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PubMed2005

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique.

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.

Journal Article↗
PubMed2001

Combined therapy with interferon and ribavirin in chronic hepatitis C does not affect serum quasispecies diversity.

Our aim was to investigate if interferon plus ribavirin has any effect on serum HCV quasispecies distribution and the relationship between diversity of HCV quasispecies and treatment response. In all, 21 patients were treated with interferon plus ribavirin for 48 weeks. The presence of HCV quasispecies was determined in serum samples at baseline and at the fourth week of treatment by SSCP analysis of the hypervariable region. SSCP pattern was defined as single or multiple band. A single band was found in six patients and multiple bands in nine. No significant difference was found between SSCP pattern in pretreatment samples and response to the therapy. In none of the patients were observed changes in number of SSCP bands between samples taken at baseline and in the fourth week of the therapy. In conclusion, the complexity of HCV quasispecies before the therapy was not related to treatment response; combined therapy did not affect serum HCV quasispecies.

Adult↗
PubMed1998

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.

Animals↗
PubMed2002

Stability of arsenic species and insoluble arsenic in human urine.

Urinary arsenic species are important short-term biomarkers that have been used in epidemiological studies. However, the stability of soluble arsenic species and the amount of arsenic lost during sample pretreatment remain unclear. The objective of this study is to evaluate the stability of soluble arsenic species in urine and aqueous standards, as well as to assess the amount of insoluble and soluble arsenic lost during pretreatment (centrifugation and filtration, respectively). High-performance liquid chromatogram inductively coupled plasma mass spectrometry was used to speciate arsenic species [Arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], and arsenobetaine [AsB]] in aqueous standards and in urine samples. The arsenic levels in both freshly collected urine samples (pH = 5.5-7.0) and National Institute of Standards and Technology Standard Reference Material 2670 toxic elements in frozen-dried urine (pH 4.4) remained constant up to 6 months when stored at -20 degrees C. In an aqueous solution mixed with 10 micro g/liter of As(III), As(V), MMA, and DMA standards, and stored at 4 degrees C, As(III) and As(V) were stable only up to 4 weeks, and MMA and DMA remained stable up to 4.5 months. The same phenomenon was observed for 100 micro g/liter mixed aqueous standards. There was no significant loss of arsenic species in urine (<5%) when passed through a 0.45- micro m filter. The amounts of insoluble arsenic in urine lost during centrifuge ranged from 1/2 to 1/17 of soluble arsenic. These findings indicated that the urinary matrix plays an important role in stabilizing arsenic species. Also, the loss of insoluble arsenic in urine during centrifuging results in underestimation of arsenic exposure, and may explain the lack of an association between arsenic exposure and the risk of health outcomes reported in some epidemiological studies.

Arsenates↗
PubMed1998

Effect of arsenosugar ingestion on urinary arsenic speciation.

We developed and evaluated a method for the determination of microgram/L concentrations of individual arsenic species in urine samples. We have mainly studied arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) because these are the most commonly used biomarkers of exposure by the general population to inorganic arsenic and because of concerns over these arsenic species on their toxicity and carcinogenicity. We have also detected five unidentified urinary arsenic species resulting from the metabolism of arsenosugars. We combined ion pair liquid chromatography with on-line hydride generation and subsequent atomic fluorescence detection (HPLC/HGAFS). Detection limits, determined as three times the standard deviation of the baseline noise, are 0.8, 1.2, 0.7, and 1.0 mu/L arsenic for arsenite, arsenate, MMAA, and DMAA, respectively. These correspond to 16, 24, 14, and 20 pg of arsenic, respectively, for a 20-muL sample injected for analysis. The excellent detection limit enabled us to determine trace concentrations of arsenic species in urine samples from healthy subjects who did not have excess exposure to arsenic. There was no need for any sample pretreatment step. We used Standard Reference Materials, containing both normal and increased concentrations of arsenic, to validate the method. Interlaboratory studies with independent techniques also confirmed the results obtained with the HPLC/HGAFS method. We demonstrated an application of the method to the determination of arsenic species in urine samples after the ingestion of seaweed by four volunteers. We observed substantial increases of DMAA concentrations in the samples collected from the volunteers after the consumption of seaweed. The increase of urinary DMAA concentration is due to the metabolism of arsenosugars that are present in the seaweed. Our results suggest that the commonly used biomarkers of exposure to inorganic arsenic, based on the measurement of arsenite, arsenate, MMAA, and DMAA, are not reliable when arsenosugars are ingested from the diet.

Arsenates↗
PubMed1995

Glutathione suppresses spontaneous activity in the frog spinal cord.

Spontaneous electrical activity in the isolated hemisected frog spinal cord increased in the presence of the SH reductant dithiothreitol (DTT) was reversibly suppressed by the oxidant 5-5'-dithio-bis-(2 nitrobenzoic acid) (DTNB) and was irreversibly suppressed by the sulphydryl modifying agents N-ethyl-maleimide (NEM) and monobromotrimethylammonio bimane. Glutathione (GSH), an important natural low molecular weight thiol, reversibly suppressed spontaneous activity. Cords pretreated with glutathione and successively exposed to NEM or bimane maintained their normal electrical activity. This indicates that GSH had interacted with the exposed sulphydryls and prevented their reaction with NEM or bimane. Incubation with bimane resulted in fluorescence-labelled neurones in the dorsal and ventral horns, whereas samples pretreated with NEM or with GSH were not labelled. Neurones appeared again fluorescent in cords preincubated with GSH and sequentially exposed to NEM or bimane. Both electrophysiological and histochemical methods indicate that exposed membrane sulphydryls are involved in the genesis and/or modulation of spontaneous electrical potentials.

Animals↗
PubMed1983

HPLC--residue analysis of the herbicide pyridate in cereals.

For many reasons residue analysis of plant protecting chemicals is becoming of increasing importance. The demand of modern trace analysis techniques is to detect significantly and sensitively values down to levels in the ppb range or even less. Aggravating circumstances are the complex multicomponent sample matrices out of whom the residue analysis has to be performed; theoretically thousands of compounds could be present and interferences with substances of interest are quite likely. One way to get around these difficulties is to employ multidimensional HPLC (MD-HPLC). A method using this invaluable analytical tool tracing Pyridate and its main metabolites in various plant extracts is presented. Based on a brief discussion about requirements of modern trace analysis HPLC in connection with column switching we designed an automatizable setup combining a weak anionexchanger (dimethylamine phase) with a reversed phase system handling relatively large aqueous sample volumes. Depending on specifically described sample pretreatment schemes detection limits down to 30 ppb are routinely obtained. At this level acceptable on-line UV-spectra can be obtained via inserting a spectrophotometric detector in a loop of a 6-port valve. The usefulness of the method described has been demonstrated by analysing several hundred samples.

Chromatography, High Pressure Liquid↗
PubMed2000

Determination of ifosfamide, 2- and 3-dechloroethyifosfamide using gas chromatography with nitrogen-phosphorus or mass spectrometry detection.

A comparison was made between methods for determining ifosfamide (IF), 2- (2DCE) and 3-dechloroethylifosfamide (3DCE) using gas chromatography with nitrogen-phosphorus detection (GC-NPD) versus positive ion electron-impact ion-trap mass spectrometry (GC-MS2). Sample pretreatment involved liquid-liquid extraction with ethyl acetate after adding trofosfamide as internal standard and alkalinization. The GC-NPD was linear, specific, and sensitive for all analytes in the range of 0.0500-100 microg/mL with lower limits of quantification (LLQ) of 0.0500 microg/mL using a 50-microgL plasma sample. The GC-MS2 was linear, specific, and sensitive for IF, 2DCE, and 3DCE in the ranges of 0.250-100, 0.500-25.0, and 0.500-25.0 microg/mL, respectively, with LLQs of 0.250, 0.500, and 0.500 microg/mL. The ranges of accuracy, within-day precision, and between-day precision for analysis of all compounds with GC-NPD did not exceed 93.3% to 105.4%, 8.0% and 9.8%, respectively. The ranges of accuracy, within-day precision, and between-day precision for analysis of all compounds with GC-MS2 did not exceed 86.5% to 99.0%, 9.0% and 12.7%, respectively. In conclusion, GC-NPD proved to be superior to GC-MS2 in sensitivity, detection range, accuracy, and precisions. Therefore GC-NPD is the method of choice for fast un-derivatized determination of IF, 2DCE, and 3DCE in human plasma, and it can readily be used for clinical pharmacokinetic studies and routine monitoring of IF-treated patients in a hospital setting.

Antineoplastic Agents, Alkylating↗
PubMed2001

Direct ultrasonic agitation for rapid extraction of organic matter from airborne particulate.

Direct ultrasonic extraction (DUE) is proposed as simple and rapid sample pretreatment method. This new approach is applied to the extraction of particulate organic matter (POM) from airborne particulate by using dichloromethane (DCM) or DCM/methanol (90/10, v/v) as extractant. The analytical determination was carried out by weighing the extractable POM on an electrobalance. Total recovery for POM could be obtained when the sample was extracted three times with 25-50 mL extractant each for about 5 min at 50 W ultrasonic power. In comparison with conventional Soxhlet extraction, less extraction time (total 15 min only) and solvent consumption (100 mL) were required by DUE. The efficiency of the DUE was similar or even higher than the routine Soxhlet method. Additionally, the new extractor is very simple and easy to use and can accelerate the extraction procedures of organic components from various solid samples.

Air↗
PubMed2005

Dynamic hollow fiber-supported headspace liquid-phase microextraction.

With the increasing concern over deteriorating environmental quality, the analysis of organic pollutants in air, water, and soil has become critically important. The development of simple, efficient, and inexpensive analytical sample pretreatment is crucial for monitoring and evaluating the environment. In this work, a dynamic hollow-fiber supported headspace liquid-phase microextraction (DHF-HS-LPME) approach was developed. In dynamic LPME, the extracting solvent is held within a hollow fiber, affixed to a syringe needle and immersed in the sample solution, and is moved to-and-fro by using a programmable syringe pump. The movement facilitates mass transfer from the sample to the solvent. Here, a similar approach was adopted, except that extraction was from the headspace rather than by direct immersion. Analysis of the extract was carried out by gas chromatography-mass spectrometry. The effect of sampling temperature, water, salt, dwelling time were investigated. Results indicated that this novel headspace microextraction method gave good analyte-enrichment factors, linear range, limits of detection and repeatability, all of which were evaluated by extracting PAHs from soil samples. This technique represents an inexpensive, convenient, fast and simple sample preparation of this class of semi-volatile organic compounds.

Chromatography, Liquid↗
PubMed2005

Cellular death mediated by nuclear factor kappa B (NF-kappaB) translocation in cultured human lens epithelial cells after ultraviolet-B irradiation.

PURPOSE: To determine the role of nuclear factor kappa B (NF-kappaB) in the death of lens epithelial cells (LECs) after ultraviolet (UV) irradiation. SETTING: Department of Ophthalmology, Ilsan Paik Hospital, Inje University, Korea. METHODS: Cultures of simian virus 40 transfected human LECs (HLE B-3 cells) were were irradiated with a UVB source (312 nm) located 10 cm from the bottom of the slides for 1, 2, 3, or 4 minutes. Cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. Translocation of NF-kappaB was examined by immunocytochemistry using anti-NF-kappaB p65 antibody and electrophoretic mobility shift assay (EMSA). Sulfasalazine, a specific NF-kappaB inhibitor, was used to confirm the role of NF-kappaB by pretreating samples for 30 minutes before UV irradiation, after which cytotoxicity and NF-kappaB translocation were evaluated. RESULTS: When HLE B-3 cells were irradiated with UVB, translocation of NF-kappaB was observed with immunocytochemistry. These translocations peaked during EMSA 6 hours after UV irradiation. In HLE B-3 cells pretreated with sulfasalazine, the translocation of NF-kappaB was blocked. Cellular death after UV irradiation was also markedly reduced by sulfasalazine pretreatment. Ultraviolet irradiation can translocate NF-kappaB, and sulfasalazine is a useful blocking agent in this pathway. In this experimental model, sulfasalazine prevented cellular death after UV irradiation. CONCLUSION: The findings suggest that NF-kappaB plays an important role in cellular death after UV irradiation.

Apoptosis↗
PubMed1989

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

Adult↗
PubMed2006

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

PURPOSE: Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors. RESULTS: Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene. CONCLUSIONS: A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.

Amino Acid Sequence↗
PubMed2004

Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction and liquid chromatography.

This study describes the determination of polycyclic aromatic hydrocarbons (PAHs) in water using high-performance liquid chromatography (HPLC) coupled with fluorescence detection (FLD). Because individual PAHs are generally present in water only at trace levels, a sensitive and accurate determination technique is essential. The separation and detection of five PAHs were run completely within 25 min by the HPLC/FLD system with an analytical C18 column, a fluorescence detection, and acetonitrile-water gradient elution. Calibration graphs were linear with very good correlation coefficients (r > 0.9998), and the detection limits were in the range of 2-6 ng/l for five PAHs. Solid phase microextraction (SPME) was performed for sample pretreatment prior to HPLC-FLD determination, and the governing parameters were investigated. Compared to conventional methods, SPME has high recovery, saves considerable time, and reduces solvents waste. The extraction efficiencies of five PAHs were above 88% and the extraction times were 35 min in one pretreatment procedure. One particular discovery is that 1.5 M sodium monochloroactate (ClCH2COONa) can improve the extraction yield of PAH compounds more than other inorganic salts. The SPME-HPLC-FLD technique provides a relatively simple, convenient, practical procedure, which was here successfully applied to determine five PAHs in water from authentic water samples.

Chemistry Techniques, Analytical↗
PubMed1992

First practical colorimetric assay of lithium in serum.

We report a colorimetric method for determining lithium in blood serum without sample pretreatment or solvent-extraction steps. The method is based on a novel chromogenic ionophore that exhibits exceptionally high selectivity (much greater than 4000:1) for lithium over sodium. The standard curve for the method is linear up to 3.5 mmol/L and exceeds the therapeutic range for lithium. The results for 57 patients' samples correlated well with results obtained with an Instrumentation Laboratory flame photometer (r = 0.97).

Colorimetry↗
PubMed1997

Microparticle-enhanced nephelometric immunoassay of lactoferrin in human milk.

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immuno-agglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675-21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%).

Colostrum↗
PubMed2006

Biological treatment of industrial wastes in a photobioreactor.

An algal-bacterial consortium was tested for the treatment from a coke factory. A Chlorella vulgaris strain and a phenol-degrading Alcaligenes sp. were first isolated from the wastewater treatment plant to serve as inocula in the subsequent biodegradation tests. Batch tests were then conducted with samples from the real wastewater or using a synthetic wastewater containing 325 mg phenol/l and 500 mg NH4+/l as target pollutants. Direct biological treatment of the real wastewater was not possible due to the toxicity of organic compounds. Activated carbon adsorption and UV(A-B)-irradiation were efficient in detoxifying the effluent for subsequent biological treatment as inoculation of pretreated samples with the algal-bacterial consortium was followed by complete phenol removal and NH4+ removal of 45%. Complete phenol removal and 33% NH4+ removal were achieved during the fed-batch treatment of artificial wastewater at 6 d hydraulic retention time (HRT). Under continuous feeding at 3.6 d HRT, phenol and NH4+ removal dropped to 58 and 18%, respectively. However, complete phenol removal and 29% NH4+ removal were achieved when 8 g NaHCO3/l was added to the artificial wastewater to enhance algal growth. This study confirms the potential of solar-based industrial wastewater treatment based on solar-based UV pretreatment followed by algal-bacterial biodegradation.

Alcaligenes↗
PubMed1997

Post-translational non-enzymatic modification of proteins. I. Chromatography of marker adducts with special emphasis to glycation reactions.

Analytical methods for marker compounds formed during post-translational modifications of proteins are reviewed. Only adducts arising either in vivo or under in vitro conditions simulating the in vivo situations are discussed. All of these compounds stem from either the reaction of free amino groups (i.e., lysine, arginine or N-terminal amino acid). In most cases the reactive counterpart is an aldehydic moiety containing endogenous compound; however, other functional groups containing metabolites are considered as well. The main demand put upon such marker compounds is that they are stable in acid or enzymatic hydrolysis or, alternatively, can be stabilized by simple sample pretreatment (e.g., by reduction). Practically all categories of separation procedures can be applied provided that the chemical characteristics of a particular marker are adequately respected; frequently combination of two different separation procedures based on different principles must be used. Because of the low level of such marker compounds under in vivo conditions, an appropriate sample enrichment step must be involved. Emphasis is put upon the analysis of Amadori products, pentosidine (and pentosidine related compounds), pyrraline, furosine, N-carboxymethylamino acids, amino acid hydantoins and stabilized dicarbonyl intermediates.

Amino Acids↗