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PubMed1995

[Reproducibility of MTD system for detection of Mycobacterium tuberculosis: a cooperative study among six laboratories].

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) is a rapid test for the detection of Mycobacterium tuberculosis, utilizing the rRNA amplification method. For assessing the reliability and reproducibility of the method, a co-operative blind study was conducted among 6 laboratories. Materials for test were sputum and water samples containing known numbers of Mycobacterium bovis BCG or Mycobacterium avium, and samples without bacteria. From three of 6 laboratories, false-positive results were reported for bacteria negative samples, however, the ratio was below 10%; 8.3% (3/36 samples), 5.6% (2/36), and 2.8% (1/36), respectively. It indicates the indispensability of negative controls for sample pretreatment and RNA extraction stages in the routine MTD test. In every laboratory, all the samples with 10(2) BCG in water and 10(4) BCG in sputum were found to be MTD positive. For the sputum samples with 10(2) BCG, positive results with the ratio above 80% were reported from 4 laboratories. These results indicate that the MTD test based on rRNA amplification method is quite useful for the rapid diagnosis of M. tuberculosis infection.

Humans↗

PubMed1996

Sodium dodecyl sulphate removal from tryptic digest samples for on-line capillary liquid chromatography/electrospray mass spectrometry.

An on-line microcolumn switching method was developed for the removal of sodium dodecyl sulphate (SDS) from tryptic digest samples. The system includes two micro-precolumns: a specific ionic detergent trapping column and a preconcentration column. Characterization of the proteinaceous samples, after isolation from the SDS, was performed by capillary liquid chromatography (LC) with UV absorption detection and electrospay mass spectrometry (ESI-MS). Loading and clean-up of the samples and regeneration of the detergent trapping column were performed at 50 microl min(-1), resulting in sample clean-up times of only 30 s. SDS-containing tryptic digested protein samples were directly applied to the micro-precolumns without any previous sample pretreatment. The developed microcolumn switching method permits the on-line analysis of small tryptic digest samples by capillary LC/ESI-MS in the presence of SDS. The method is completely automated and can be performed unattended. The maximum amount of SDS, in terms of loadability and breakthrough, were determined. Also studied were the selection of the loading and clean-up solvents and the recovery of the peptides. Chromatographic separations and mass spectral data confirmed the removal of SDS.

Amino Acid Sequence↗

PubMed2005

Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection.

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.

Acids↗

PubMed2006

On-line derivatization gas chromatography with furan chemical ionization tandem mass spectrometry for screening of amphetamines in urine.

A simple alternative method with minimal sample pretreatment is investigated for screening of amphetamines in small volume (using only 20 microL) of urine sample. The method is sensitive and selective. The method uses gas chromatography (GC) direct sample introduction (DSI) for on-line derivatization (acylation) of amphetamines to improve sensitivity. Furan as chemical ionization (CI) reagent in conjunction with tandem mass spectrometry (MS/MS) is used to improve selectivity. Low background with sharp protonated molecular ion peaks of analytes is the evidence of improvement in sensitivity and selectivity. Blank urine samples spiked with known amounts of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine is analyzed. Selected ion monitoring of the characteristic product ions (m/z 119+136+150+163) using furan CI-MS/MS in positive ion mode is used for quantification. Limits of detection (LOD) between 0.4 and 1.0 ng mL(-1) and limits of quantitation (LOQ) between 1.0 and 2.0 ng mL(-1) are established. Linear response over the range of 1-1000 ng mL(-1) (r(2)>0.997) is observed for all analytes, except for methamphetamine (2.0-1000 ng mL(-1)). Good accuracy between 86 and 113% and precision ranging from 4 to 18% is obtained. The method is also tested on real samples of urine from suspected drug abusers. This method could be used for screening and determination of amphetamines in urine samples, however needs additional work for full validation.

Amphetamines↗

PubMed2003

Simultaneous determination of neutral and acidic pharmaceuticals in wastewater by high-performance liquid chromatography-post-column photochemically induced fluorimetry.

An analytical method for the simultaneous determination of acidic and neutral pharmaceutical active compound (PhACs) residues in wastewater has been developed based on the combination of high-performance liquid chromatography (HPLC) and photochemically induced fluorimetry. The photoderivatization conditions for each particular PhAC have been assessed. Off-line optimization of the HPLC separation for both neutral and acidic compounds has been utilised and evaluated. Detection limits in the low ng/ml range have been achieved without sample pretreatment. By applying the developed analytical method combined with solid-phase extraction to real wastewater samples an enrichment factor of approximately two orders of magnitude can be obtained.

Chromatography, High Pressure Liquid↗

PubMed2002

Improved separation of microheterogeneities and isoforms of proteins by capillary electrophoresis using segmental filling with SDS and PEO in the background electrolyte.

To improve the separation efficiency while achieving high sensitivity for the analysis of proteins' microheterogeneity, a segmental-filling technique has been developed and tested in capillary electrophoresis with laser-induced native fluorescence using a pulsed Nd:YAG laser. Using a short plug of SDS applied to the capillary and the anticonvectant poly(ethylene oxide) (PEO), the microheterogeneities of a number of proteins with pI values ranging from 4.5 to 11.1 could be detected. This high resolving power is due to reduced adsorption on the capillary wall, sieving, and the interaction with SDS. Consequently, the length and the concentration of the SDS plug play a significant role in determining the resolution and sensitivity. The method has been applied to the analysis of salivary and cerebrospinal fluid (CSF) samples. Without any sample pretreatment, using a 10-s 1x SDS plug, six alpha-amylase isoforms in a salivary sample were resolved in 17 min and three more peaks were detected in a CSF sample. With simplicity, high resolving power, and rapidity, the method has shown great potential for proteomics.

Electrolytes↗

PubMed1999

A modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA) to determine cyclosporin A concentrations in rat skin.

We have developed a simple, sensitive and reliable assay procedure for cyclosporin A (CyA), a modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA), to determine the CyA content in rat skin. The conventional fluorescence polarization immunoassay (FPIA) method for CyA using a commercially available FPIA kit, TDX cyclosporine monoclonal whole blood, was modified. A fat emulsion for intravenous infusion, Intralipos, was incorporated for dissolving the CyA extracted from the skin tissue, and a mixture of MeOH/purified water was used as the sample pretreatment medium instead of the precipitation reagent in the conventional FPIA kit intended for whole blood samples. These modifications enabled us to produce a reliable and the sensitive assay of CyA in skin tissue. The reproducibility (coefficient of variation), detection limit, and assay time for FE-FPIA were below 2%, 25 ng/ml, and about 24 min/24 samples, respectively, and were comparable with those for the whole blood samples determined by the conventional FPIA. Pre-purification of samples required by the HPLC assay is not needed in the FE-FPIA method. The usefulness of the FE-FPIA method in evaluating the topical pharmacokinetics of CyA in skin is discussed.

Animals↗

PubMed2004

Mercury fractionation in contaminated soils from the Idrija mercury mine region.

Mercury (Hg) fractionation was investigated in contaminated soil in the Idrija Hg-mine region, Slovenia. The main aim of this study was to test and apply sequential extraction and quantification of different Hg phases in order to estimate the mobility and potential bioavailability of Hg in contaminated soils. Separation of Hg phases was performed by means of a selective sequential extraction procedure complemented by volatilization of elemental mercury (Hg0). The influence of temperature, moisture and storage on Hg0 volatilization was also investigated. The total Hg concentrations varied between 8.4 and 415 mg kg(-1) and were up to 40-fold higher than the maximum permissible set by Slovenian legislation. Fractionation measurements indicated cinnabar as the predominant Hg fraction, followed by Hg0. Accumulation of cinnabar predominantly occurred in coarse grained flood plain sediments, where on average it constituted more than 80% of total Hg. In contrast non-cinnabar fractions were found to be enriched in areas where fine grained material was deposited, reaching up to 60% of total Hg. The strong positive correlation (R2 = 0.71-0.99) among non-cinnabar fractions suggested that these fractions predominantly control the mobility and potential bioavailability of Hg. Sample pretreatment before fractionation influenced the partition of Hg between different fractions, and therefore fractionation in fresh, nontreated samples is suggested. In addition, the specificity of the extraction steps needs further attention, as it was shown that some extraction steps, such as the organo-chelating Hg fraction, do not provide meaningful results. This further suggests that protocols for mercury fractionation need further harmonization in order to improve the comparability of the results and their use in risk assessment. Volatile mercury fluxes averaged between 0.04 and 6.5 ng g(-1) h(-1). Good agreement (R2 = 0.81-0.95) was found between the non-cinnabar fractions and evaporation of Hg0. Both the temperature and sample moisture had significant effects on mercury volatilization. The results in this study were obtained at 70 degrees C, which may be somewhat high, in particular for bacterial activity which may also play an important role in Hg volatilization. Therefore it is strongly suggested that further optimisation of the protocol to assess Hg volatilization from soil is required.

Chemical Fractionation↗

PubMed1976

Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples.

For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.

Adsorption↗

PubMed2005

Low-temperature reaction of trialkoxysilanes on silica gel: a mild and controlled method for modifying silica surfaces.

The room temperature reaction of 4-(triethoxysilyl)butyronitrile, 4-TBN ((C2H5O)3Si(CH2)3CN), on weakly hydrated silica samples pretreated at 393 K has been studied by desorption experiments and by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy at different aging times under various water partial pressures. The reaction is demonstrated by the decrease of desorption of 4-TBN with time and the simultaneous disappearance of the 2980 and 1394 cm(-1) signals in the DRIFT spectra, assigned to the CH3 moiety of the ethoxy functions. Water partial pressure is shown to have a crucial effect on the rate and efficiency of the process as, after 6 days, for samples kept at room temperature under vacuum, ca. 50% of the silane has reacted, while for those kept in a water-saturated atmosphere the silane reaction reaches 96%. Although the silane appears to be irreversibly bonded to the surface, no definite conclusion may be drawn from these preliminary results as to the nature of the bonding (grafting or coating). These samples are compared to modified silicas prepared according to conventional methods. The same extent of silane reaction (50%) is achieved for preadsorbed samples kept under vacuum and either cured at 473 K for 30 h or kept at room temperature for 6 days. A mild and controlled modification of silica by triethoxysilanes can thus be achieved by first physisorbing known amounts of the modifying silanes from an organic solvent on pretreated silica and then letting the samples mature for a few days at room temperature in a water-saturated atmosphere.

Journal Article↗

PubMed2000

Stability of polymorphic forms of ranitidine hydrochloride.

Ranitidine-HCl can exist in two different polymorphic forms: form I (m.p. 134-140 degrees C) and form II (m.p. 140-144 degrees C). In the present study the stability of form I of ranitidine-HCl to a selection of powder pretreatments, to reflect conditions which might occur in manufacturing procedures, and also to a limited range of storage conditions was investigated. The original samples of form I and form II used were characterised by X-ray powder diffraction (XRPD), hot stage microscopy (HSM) and differential scanning calorimetry (DSC). A quantitative XRPD method for determining the fraction of form II in the presence of form I was used. XRPD data were analysed using regression techniques and artificial neural networks (ANN). The quantitative XRPD technique was then used to monitor the relative proportion of form II in each treated sample. Pretreatments of form I included (i) mixing with form II or with common excipients (ii) compression and grinding (iii) contact with solvents (followed by drying) before storage. Storage conditions involved three temperatures (20 degrees C, 30 degrees C, 42 degrees C) and three relative humidities (45% RH; 55% RH; 75% RH). Samples were stored for a period of 6 months. A limited factorial design was used. No increase in the form II:form I ratio was observed in the following pretreatment processes: introduction of form II nuclei into form I; introduction of excipients to form I; compression of form I powder at 5 and 15 tons; normal mixing and grinding processes; addition of isopropanol (IPA) or water/IPA mix followed by drying. In the pretreatment process where water was added to form I powder (with most or all of the powder dissolving), drying of the liquefied mass led to a mix of form I and form II. On storage at room temperature (20-30 degrees C), low relative humidity (45-55% RH), and in an air-tight container there was no increase in the form II:form I ratio. Storage of form I/form II mixes, particularly at high humidity, resulted in a preferential loss of form II (compared to form I). Loss was greater at 30 degrees C/75% RH than at 20 degrees C/75% RH. Form II was also preferentially lost under low humidity conditions created by a saturated solution of potassium carbonate (45% RH) at the elevated temperature of 42 degrees C. This environment was shown to be acidic.

Calorimetry, Differential Scanning↗

PubMed1994

Simultaneous determination of urinary creatinine, calcium and other inorganic cations by capillary zone electrophoresis with indirect ultraviolet detection.

A method for the rapid analysis of calcium and creatinine in urine has been developed using capillary zone electrophoresis without sample pretreatment other than dilution. Cations of NH3, K, Na, Mg, Ca, Li, Ba and creatinine in human urine samples could be separated within 7 min. Background electrolyte (BGE) was composed of 5 mmol/l pyridine (chromophore), 3.6 mmol/l tartaric acid and 2 mmol/l 18-crown-6, having a pH of 4.05. An indirect UV detection mode was employed at 255 nm. Efforts were made to eliminate the interaction between cations and proteins and to minimize the electromigrative dispersion (EMD) for the analytes of interest by selecting a suitable BGE and sample dilution solution. Quantitative analyses were performed for calcium and creatinine. The calibration plots showed good linearity over the concentration range of interest to clinical analysis. Data on recoveries and reproducibilities are also reported. Results for urine samples (previously collected and frozen) from a variety of healthy and pathological individuals were in good agreement with those obtained by the Technicon SMA II calcium and creatinine methods.

Ammonia↗

PubMed1997

High-performance liquid chromatographic assay using electrochemical detection for the combined measurement of amifostine, WR 1065 and the disulfides in plasma.

A high-performance liquid chromatographic (HPLC) method was developed for the combined analysis of the chemoprotective agent, amifostine, its active metabolite, WR 1065, and the (symmetrical and mixed) disulfides of WR 1065 in plasma. These three compounds were quantified by measuring WR 1065 after three different sample pretreatment procedures. During these procedures, amifostine and the disulfides were quantitatively converted into WR 1065, by incubating the sample either at a low pH or in the presence of dithiothreitol, respectively. The resulting amounts of WR 1065 were determined by HPLC with electrochemical detection (Au electrode, + 1.00 V). The lower limit of quantitation of WR 1065 was 0.15 microM. The within-day and between-day precision were < or =4.4 and < or =8.2% for WR 1065, < or =4.9 and < or =13.1% for amifostine and < or =8.5 and < or =5.5% for the disulfides, respectively. The within-day and between-day accuracy ranged from 97.2 to 109.8% and from 97.6 to 101.5% for WR 1065, from 88.3 to 110.7% and from 99.4 to 101.5% for amifostine and from 99.2 to 110.2% and from 103.3 to 104.9% for the disulfides, respectively. This method is superior to other described methods due to its simple and relatively rapid analysis of all three compounds in one system. Furthermore, it is at least as sensitive as earlier reported methods for one of the compounds and the application of the gold electrode requires only minor maintenance. Therefore, this method is very suitable for pharmacokinetic studies of amifostine and its metabolites. As an example, the plasma concentrations of amifostine, WR 1065 and the disulfides are shown in a patient after receiving an i.v. dose of 740 mg/m2 amifostine.

Amifostine↗

PubMed1999

Simultaneous determination of trace benzodiazepines from drinks by using direct electrospray probe/mass spectrometry (DEP/MS).

This study presents a novel means of rapidly determining benzodiazepines in various drinks. Electrospray mass spectrometry with a direct probe design is used as the analytical instrument. Samples are treated only by a simple liquid-liquid extraction prior to direct electrospray probe/mass spectrometry (DEP/MS) analysis. The proposed method provides a relatively easy and efficient means of identifying the drugs in a very short time interval. On average, sample analysis is completed in less than five minutes including sample pretreatment. It is especially useful in forensic science since the technology can provide a rapid identification for unknown samples. Thus, the method proposed herein is highly appropriate for rapidly screening a large number of samples.

Beer↗

PubMed1999

Determination of vitamin D3 metabolites: state-of-the-art and trends.

The steps involved in the methods for the determination of vitamin D3 metabolites (namely, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3) mainly in clinical samples are critically reviewed. Sample pretreatment (e.g. deproteinization, saponification, liquid liquid and liquid solid extraction, etc.) as a function of both type of sample and detection system, quantitation based on protein saturation and liquid as well as gas chromatography are discussed. The chemical principles on which the methods are based and the derivatization procedures, which facilitate separation and/or detection, are also commented upon. Finally, the future prospects of the research on methods for the determination of these metabolites are outlined.

24,25-Dihydroxyvitamin D 3↗

PubMed2002

Hollow fiber-protected liquid-phase microextraction of triazine herbicides.

A new microextraction technique termed hollow fiber-protected liquid-phase microextraction (LPME) was developed. Triazines were employed as model compounds to assess the extraction procedure and were determined by gas chromatography/mass spectrometry. Toluene functioned as both the extraction solvent and the impregnation solvent. Some important extraction parameters, such as effect of salt, agitation, pH, and exposure time were optimized. The new method provided good average enrichment factors of > 150 for eight analytes, good repeatability (RSDs <3.50%, n = 7), and good linearity (r2 > or = 0.9995) for spiked deionized water samples. The limits of detection (LODs) were in the range of 0.007-0.063 microg/L (S/N = 3) under selected ion monitoring mode. In addition to enrichment, hollow fiber-protected LPME also served as a technique for sample cleanup because of the selectivity of the membrane, which prevented large molecules and extraneous materials, such as humic acids in solution, from being extracted. The utilization of this procedure in the extraction of a slurry sample (mixture of soil and water) also gave good precision (RSDs <5.00%, n = 3) and LODs (0.04-0.18 microg/L, S/N = 3). Finally, the comparison of the new method with the static solvent drop LPME and solid-phase microextraction was performed. The results demonstrated that hollow fiber-protected LPME was a fast, accurate, and stable sample pretreatment method that gave very good enrichment factors for the extraction of triazine herbicides from aqueous or slurry samples.

Gas Chromatography-Mass Spectrometry↗

PubMed2000

Direct coupling of microcolumn liquid chromatography with in-tube solid-phase microextraction for the analysis of antidepressant drugs.

The direct coupling of in-tube solid-phase microextraction (in-tube SPME) with microcolumn liquid chromatography (micro-LC) has been investigated for the analysis of antidepressants in human urine. The use of in-tube SPME has been clearly shown to be advantageous for the on-line coupling of the SPME method, as the sample pretreatment technique, with micro-LC as the separation technique. This is because much smaller amounts of the sample solutions, desorption solvents and the mobile phase are required compared with conventional SPME-LC systems. The parameters for preconcentration have been investigated for the extraction capillary with the newly developed 'wire-in-tube' configuration.

Antidepressive Agents↗

PubMed2001

Investigation of bacteremia after orthodontic banding and debanding following chlorhexidine mouth wash application.

This study investigates the prevalence of bacteremia after orthodontic banding and debanding, following the application of a 0.2% chlorhexidine gluconate mouthwash. The banding and debanding groups were each composed of 40 young adult patients. In the banding group, patients were asked to rinse their mouth with chlorhexidine gluconate for 60 seconds just prior to fitting of the bands. In the debanding group, they were asked to use the mouthwash immediately before removal of bands and brackets. In both groups pre- and post-treatment blood samples were obtained with a strict aseptic technique. In the banding group, no bacteremia was detected in the pretreatment sample and 2.5% post-treatment bacteremia was detected in the post-treatment sample. In the debanding group, 2.5% bacteremia was found in both the pre- and post-treatment samples. The prevalence of post-treatment bacteremia found in the present study were compared with the findings of 2 preliminary studies in which the prevalence of bacteremia had been investigated after banding and debanding without a prior application of chlorhexidine mouthwash. The application of chlorhexidine mouthwash resulted in a decrease in the prevalence of bacteremia after banding and debanding, but the decrease was not statistically significant.

Adolescent↗