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PubMed2004

Chemometric determination of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate in synthetic mixtures and pharmaceutical formulations.

Different chemometric methods such as classical least squares (CLS), principal components regression (PCR) and partial least squares with one dependent variable (PLS-1) applied on UV spectral data (0 D) and on their first derivatives (1 D) were evaluated for the simultaneous quantification of samples containing mixtures of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate. Their performances were compared by means of ANOVA tests, which evidenced that 0 D-PCR, 0D-PLS-1, 1D-PCR, 1D-PLS-1, were reproducible and gave statistically similar results, while 0 D-CLS and 1D-CLS displayed higher variances than the former and failed to comply with the Levene's variance homogeneity test at different stages of the method comparison and validation process. The four statistically equivalent procedures were successfully applied to the analysis of synthetic samples with two to four analytes and to commercial tablet preparations containing amiloride hydrochloride and hydrochlorothiazide alone or in association with atenolol or timolol maleate.

Amiloride↗
PubMed2006

Biosensing systems for the detection of bacterial quorum signaling molecules.

Bacterial quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of certain specialized genes depending on their cell population size. Signaling molecules such N-acylhomoserine lactones (AHLs) mediate the communication, and their concentration reflects the bacterial population density. Quorum sensing regulates several processes including bacterial pathogenicity. We developed a method for the rapid, sensitive, and quantitative detection of AHLs in biological samples such as saliva and stools. The method is based on whole-cell sensing systems that employ QS regulatory systems as recognition elements and the luxCDABE gene cassette as a reporter. The method proved to be reproducible when applied to real samples and was able to detect low analyte concentrations down to 1 x 10(-9) M without requiring extensive sample preparation. We envision that this novel biosensing system could be employed in the diagnosis and management of various bacteria-related disorders, thus supporting the use of quorum sensing molecules as potential biomarkers of disease. Due to cost-effectiveness and high throughput, these biosensing systems could be successfully employed as a new tool for the screening of novel drugs that target quorum sensing mechanisms.

Bacteria↗
PubMed2005

Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatography-electrospray tandem mass spectrometry.

A rapid multiresidue method to quantify three different classes of plant hormones has been developed. The reduced concentrations of these metabolites in real samples with complex matrixes require sensitive techniques for their quantification in small amounts of plant tissue. The method described combines high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Deuterium-labeled standards were added prior to sample extraction to achieve an accurate quantification of abscisic acid, indole-3-acetic acid, and jasmonic acid in a single run. A simple method of extraction and purification involving only centrifugation, a partition against diethyl ether, and filtration was developed and the analytical method validated in four different plant tissues, citrus leaves, papaya roots, barley seedlings, and barley immature embryos. This method represents a clear advantage because it extensively reduces sample preparation and total time for routine analysis of phytohormones in real plant samples.

Carrier Proteins↗
PubMed2005

Two fish bile reference materials certified for PAH metabolites.

Environmental pollution with polycyclic aromatic hydrocarbons (PAHs) from pyrogenic or petrochemical sources is a matter of concern in many aquatic ecosystems. For field monitoring purposes, PAH metabolite levels in fish bile samples can be used as biomarkers of exposure, but previously there were no reference materials for analytical method validation or quality control. In this paper we describe the preparation and certification of two fish bile materials. BCR 720 is a pure bile material from sediment-exposed flounder (Platichthys flesus) and BCR 721 is a pure bile material from oil-exposed plaice (Pleuronectes platessa). Homogeneity and stability tests were carried out and a group of 12 expert laboratories was assembled for the certification measurements. The methods applied were liquid chromatography with fluorescence detection or gas chromatography with mass spectrometric detection. In BCR 720 three PAH metabolites were certified: 1-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene. In BCR 721 1-hydroxypyrene was certified. The fourth target compound, 2-hydroxynaphthalene, could not be certified, but the results obtained are listed. The major findings from earlier feasibility studies and recommendations for the optimal use of the reference materials are also included.

Animals↗
PubMed1991

[Substance abuse analysis of biological materials].

Requests are increasing for biomedical analyses of abused drugs in forensic toxicology, tests at work, testing of drug addicts during rehabilitation, and in therapeutic and other situations. Special guidelines have been prepared in forensic toxicology, with regard to the legal background, information on any sanctions in the event of positive test results, chains of custody for handling samples, analytical methods with known specificity and sensitivity, and interpretation of results based on information of legal drug use. These guidelines should also be recommended in other cases if the results might lead to negative sanctions for the individuals involved. False positive test results have been recorded from cases analysed at local laboratories which had not established the recommended guidelines. The most critical point appears to be the confirmation of positive screening results using specific methods.

False Negative Reactions↗
PubMedDate not supplied

Variability associated with analytical methods used to measure aflatoxin in agricultural commodities.

A total of 1019 analytical precision estimates obtained from method-performance (collaborative) studies for mycotoxins published through 1991 were sorted by type of variance measurement, type of analytical method, and type of agricultural commodity. Precision estimates for total aflatoxin were sorted into 2 precision measurements (among-laboratories and within-laboratory), 3 analytical methods (thin-layer chromatography [TLC], liquid chromatography [LC], and enzyme-linked immunosorbent assay [ELISA]), and 11 agricultural commodities. Sufficient data existed to study the analytical variability (precision) associated with 36 sorted combinations (of a possible 66). In all but one combination (within-laboratory, barley, and TLC), the variance (V) was a function of total aflatoxin concentration (C). A power function of the form V = aCb, where a and b are constants, describes the relationship between variance and aflatoxin concentration. The coefficients a and b were determined from regression analysis. When results were pooled across all agricultural commodities, LC had the lowest analytical variability while ELISA had the highest. For a given method, among-laboratories variability was approximately double the within-laboratory variability. These analytical variability estimates can be coupled with previously determined variability estimates of sampling and sample preparation to determine the performance associated with specific test procedures used to inspect agricultural commodities for aflatoxin.

Aflatoxins↗
PubMed2006

Rapid column-switching liquid chromatography/mass spectrometric assay for DHEA-sulfate in the plasma of patients with Alzheimer's disease.

A simple and highly sensitive method for the quantification of dehydroepiandrosterone-3-sulfate (DHEAS) in human plasma was developed. DHEAS was directly determined in plasma using column-switching liquid chromatography/mass spectrometry (LC-MS). The plasma was filtered with a membrane filter. The filtrate was injected onto a pre-column without further sample preparation such as extraction or derivatization. The pre-column was washed with an aqueous solution to remove interference and the analyte was eluted into a reversed-phase C(18) analytical column for separation and detection using a column-switching valve. The calibration range of DHEAS was 0.01-10 micromol/L, and the linearity of the method was 0.999. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 was 5 nmol/L. The accuracy and precision (%CV) were less than 10% in within-day and day-to-day variations. To explore the relationship between Alzheimer's disease and the DHEAS level in human plasma, the concentrations of DHEAS in female patients with Alzheimer's disease (n = 20) and in normal female subjects (n = 20) were measured. The level of DHEAS was significantly decreased in the plasma of patients with Alzheimer's disease (p < 0.0002) compared with that in normal subjects. From the results, we concluded that our method is sufficiently sensitivity and reliability for the quantification of DHEAS in clinical samples. Plasma DHEAS concentration could be an important marker to understand the pathogenesis of Alzheimer's disease.

Aged↗
PubMed1993

Development and validation of an assay to measure bioactivity of human calcitonin in vitro using T47D cell membranes.

We have developed a "test-tube" assay to determine the biological activity of synthetic preparations of human calcitonin (hCT). The method is based on the dose-dependent accumulation of cyclic AMP in cell membrane preparations on stimulation with CT. As an unlimited cell source, we used the clonal cell line T47D, which possesses functional CT receptor-adenylate cyclase complexes. Half-maximal stimulation was achieved with 40-200 nmol/liter hCT. The method was able to precisely quantify the relative potencies of various hCT preparations. The intraassay and interassay coefficients of variation were 3.0 and 5.6%, respectively. The analytical performance, as estimated by additional recovery and specificity studies, was better than or comparable to the established in vivo rat hypocalcemia assay. Results obtained with these two methods were closely correlated (r = 0.83, P < 0.01). However, the in vitro membrane system allowed a higher throughput of samples, less preparation time, and better standardization and transportability of the assay, as large-scale membrane preparations were stable for at least 12 months in liquid nitrogen. This new in vitro membrane bioassay provides a convenient alternative to the currently performed in vivo rat hypocalcemia bioassays.

Animals↗
PubMed2005

Comprehensive pore structure characterization of silica monoliths with controlled mesopore size and macropore size by nitrogen sorption, mercury porosimetry, transmission electron microscopy and inverse size exclusion chromatography.

The porosity of monolithic silica columns is measured by using different analytical methods. Two sets of monoliths were prepared with a given mesopore diameter of 10 and 25 nm, respectively and with gradated macropore diameters between 1.8 and 7.5 microm. After preparing the two sets of monolithic silica columns with different macro- and mesopores the internal, external and total porosity of these columns are determined by inverse size-exclusion chromatography (ISEC) using polystyrene samples of narrow molecular size distribution and known average molecular weight. The ISEC data from the 4.6 mm analytical monolithic silica columns are used to determine the structural properties of monolithic silica capillaries (100 microm I.D.) prepared as a third set of samples. The ISEC results illustrate a multimodal mesopore structure (mesopores are pores with stagnant zones) of the monoliths. It is found by ISEC that the ratio of the different types of pores is dependent on the change in diameter of the macropores (serve as flow-through pores). The porosity data achieved from the mercury penetration measurement and nitrogen adsorption as well of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) pictures are correlated with the results we calculated from the ISEC measurements. The ISEC results, namely the multimodal pore structure of the monoliths, reported in several publications, are not confirmed analyzing the pore structures of the different silica monoliths using all other analytical methods.

Adsorption↗
PubMed2005

Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC.

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.

Animals↗
PubMed1998

Direct determination of urinary iodine by inductively coupled plasma mass spectrometry using isotope dilution with iodine-129.

An inductively coupled mass spectrometric method was developed for the direct determination of iodine in urine. The application of isotope dilution analysis with added 129I offers new possibilities for automatic and accurate determinations. The sample preparation consists of dilution with an ammonia solution containing 129I. The validation was made by comparison with the results obtained in another laboratory by a spectrophotometric method based on the Sandell-Kolthoff reaction. Different regression models, including maximum likelihood estimation, were used to compare the methods. None of the models revealed analytical bias between the two methods. The urine samples analyzed for validation were from three persons previously exposed to an iodine bath and covered a concentration range of 0.2 to 2.8 micromol/L. A detection limit of 0.02 micromol/L, a within-run CV of 2.5%, and a between-run CV of 11.9% were estimated for the proposed method.

Adult↗
PubMed2005

Chiral stability of synthetic pyrethroid insecticides.

Synthetic pyrethroids are chiral compounds consisting of multiple stereoisomers. Evaluation of enantioselectivity in environmental fate and ecotoxicity requires analytical methods that preserve stereoisomer integrity during analysis. In this study, we characterized the stability of stereoisomers from four commonly used pyrethroids, cis-bifenthrin (cis-BF), permethrin (PM), cypermethrin (CP), and cyfluthrin (CF), during gas chromatography (GC) analysis and sample preparation. Stereoisomers of cis-BF and PM were found to be stable, but those of CP and CF were unstable, under heat or in water. Isomer conversion occurred only at the alphaC in CP or CF, causing the analyte stereoisomer to convert to an epimer. At a GC inlet temperature of 260 degrees C, about 9% conversion occurred for CP and CF. In organic solvents and sterile water, stereoisomers of cis-BF and PM were stable, but slow isomer conversion was observed for CP and CF in water at ambient temperature. However, isomer conversion for CP and CF was relatively insignificant (2-3%) when the GC inlet temperature was kept at < or = 180 degrees C or when on-column injection was used. Isomer conversion at the alphaC in water suggests that abiotic processes may also contribute to enantioselectivity observed in the environment for pyrethroids with the asymmetric alphaC.

Chromatography, Gas↗
PubMed2005

Quantitative determination of the calcium channel antagonists amlodipine, lercanidipine, nitrendipine, felodipine, and lacidipine in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometric method has been developed and validated for the quantification of the five 1,4-dihydropyridine calcium channel antagonists amlodipine, lercanidipine, nitrendipine, felodipine, and lacidipine in human plasma. Sample preparation involved solid-phase extraction on RP-C18 cartridges with good recovery for all the compounds. Sample analysis was performed on a Luna RP-C18 analytical column (15 mm x 2 mm ID, 3.0 microm) with a Sciex API 365 triple quadrupole mass spectrometer with turboionspray source and multiple reaction monitoring. The method is sensitive with a limit of detection below 1 ng/mL for each drug in plasma, with good linearity (r(2) > 0.998), over the therapeutic concentration range (1 to 40 ng/mL). All the validation data, such as accuracy, precision, and interday repeatability, were within the required limits. The method can be used for pharmacokinetic studies and therapeutic drug monitoring of the compounds in humans.

Calcium Channel Blockers↗
PubMed2001

Potential of microbore HPLC within a multiresidue method for the trace analysis of plant-protective substances in water.

The performance of microbore HPLC as a "measurement channel" within a true multiclass/multiresidue method for monitoring plant protectants in raw and potable water is demonstrated. The method has a modular design and consists of a non-selective sampling and preparation line generating 250 microL of an "extract" from a 100-mL water sample; this extract can be introduced to up to four measurement channels, as required by the analytical task. The microbore HPLC channel can be used to quantify 34 plant protectants in the 0.1 microg L(-1) concentration range by use of diode-array detection at seven different wavelengths. A solvent change is necessary to link sample preparation to microbore HPLC; this uses 50 microL of the "extract" and is accomplished directly in an autosampler vial. Performance characteristics were evaluated for tap water spiked at 0.2 microg L(-1). Average recoveries were between 65 and 100% and method detection limits were 0.07 microg L(-1) or better. The ability to provide comparable and accurate results was proven by participation in an interlaboratory comparison trial. The procedure for preparing microbore columns from 750 microm i.d. PEEK tubing is described in detail to enable the reader to prepare his own columns. The reproducibility of this preparation procedure was proven by an analysis-of-variance test.

Calibration↗
PubMed2005

Determination of nonsteroidal antiinflammatory drugs in water samples using liquid chromatography coupled with diode-array detector and mass spectrometry.

An analytical method for the determination of trace levels of six different nonsteroidal antiinflammatory drugs (NSAIDs) in water samples has been developed and validated. Environmentally relevant pharmaceuticals were chosen according to human consumption in Poland. Final analysis of the target compounds was performed by RP LC-diode-array detection-MS, whereas sample preparation included an SPE step. For this SPE step, a number of packing materials, such as LiChrolut RP-18, calixarene, Strata-X, BAKERBOND Narc-2, BAKERBOND Polar Plus, BAKERBOND styrene divinylbenzene-1, and Discovery DSC-18, were used, and their respective advantages and disadvantages in this study were discussed. The RP-18 phase was found to be the most retentive for all analytes. The detection limits for compounds in surface waters were varied from 0.005 for diflunisal to 0.095 microg/L for ibuprofen. The average recoveries of NSAIDs from the surface water samples ranged from 80 up to 103%. RSD value is relatively low (from 4% for fenoprofen up to 8% for ibuprofen). The performance of the method was tested with several environmental water samples.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2003

[Studies on the analytical method for epichlorohydrin from internal can coatings].

An improved migration test was developed for determination of trace amounts of epichlorohydrin from internal can coatings. Eight kinds of sample cans, coated mainly with epoxy resin, for foods and beverages were prepared, and both their bodies and lids were tested for migration as follows. A body was filled with n-pentane and soaked for 2 hours at 25 degrees C. A lid was soaked in n-pentane (2 mL/cm2) for 2 hours at 25 degrees C. The test solution was analyzed by GC-FID and GC/MS using two DB-WAX capillary columns with different inside diameters. The limits of quantitation were 0.05 microgram/mL by GC-FID and 0.02 microgram/mL by GC/MS with selected ion monitoring. Recoveries of spiked epichlorohydrin were 99.9-104.5% at the level of 0.05 microgram/mL and 0.5 microgram/mL, with high precision. In this study, no epichlorohydrin was found to have migrated from any of the bodies and lids.

Chromatography, Gas↗
PubMed2004

Multicomponent screening for drugs of abuse: direct analysis of urine by LC-MS-MS.

By allowing for direct injection of urine, liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical ionization has proven to be useful for analysis of drugs of abuse in human urine. The purpose of this study was to evaluate this technique by direct screening for 23 different substances (phenylethylamines, hypnotics, and N-benzylpiperazine) in urine samples. It was possible to achieve lower detection limits compared with commercial immunochemical methods. There was a linear response for all analytes with an intraday coefficient of variation of about 16%, and the gradient elution gave a variability in relative retention time of about 1%. Positive results were confirmed by reanalysis including sample preparation by solid-phase extraction. Among the 529 authentic urine samples analyzed, 35 samples were screened positive for phenylethylamines, and 20 for hypnotics. Of these, 23 (66%) samples were confirmed to be positive for phenylethylamines, and 11 (55%) for hypnotics. This study demonstrates that LC-MS-MS is a valuable complement to immunochemical screening analysis, especially for substances for which immunochemical methods are not yet available or when an increased sensitivity is needed.

Chromatography, Liquid↗
PubMed1999

Improved method for the storage of groundwater samples containing volatile organic analytes

The sorption of volatile organic analytes from water samples by the Teflon septum surface used with standard glass 40-ml sample collection vials was investigated. Analytes tested included alkanes, isoalkanes, olefins, cycloalkanes, a cycloalkene, monoaromatics, a polynuclear aromatic, and two chloroethenes. Both laboratory prepared test mix solutions and petroleum contaminated groundwater from three field sites were tested. A rapid loss of n-alkane and isoalkane concentrations (>10%) was observed within 24 h when stored at room temperature. Aliphatic losses were also observed (>10%) over a 21-day holding period when samples were held at 4 degrees C. Loss of the less sorptive analytes was demonstrated by exposing analyte solutions to greater Teflon surface areas. The demonstrated sorption of aliphatics from water samples by Teflon-lined septa indicates that the accuracy of volatile petroleum hydrocarbon determinations may be reduced by the traditional storage method. An alternative storage protocol is reported combining a lead foil septum surface and 1% (w/w) tribasic sodium phosphate dodecahydrate (Na3PO4. 12H2O) preservative. This method prevented loss of the test analytes, including alkanes and isoalkanes for at least 21 days at room temperature.

Journal Article↗