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PubMedDate not supplied

Coupling of TLC and UV-measurement for quantification of naproxen and its main metabolite in urine.

A simple sensitive method of high specificity and selectivity for quantitative determination of the non-steroidal anti-inflammatory drug naproxen and its main metabolite, 6-demethylated derivative, in biological specimens is described. Like naproxen, its metabolite absorbs maximally at 232 nm; this makes their simultaneous quantification, via direct UV-measurements at lambda max, in biological fluids quite impossible. Simple TLC-separation on silica gel F254 using chloroform + methanol (85:15, v/v) achieved the best fractionation of the unchanged drug and its metabolite from the matrix-contents of urine. UV-quantification of fractionated components could reach concentration levels of 0.2-3.0 micrograms ml-1 (ppm) in worked up urine samples. Varying levels of unchanged antiinflammatory drug and the phenolic metabolite could be accurately traced in urine samples following a 2.9 mg/kg oral dose after different time-intervals. Synthetic preparation of the metabolite by demethylation of naproxen is briefly mentioned.

Chromatography, Thin Layer↗
PubMed2000

Comparative quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human skin in vivo.

OBJECTIVE AND DESIGN: Ultraviolet (UV) exposure induces local immunosuppression and inflammation in human skin. Cytokines are, in part, responsible for these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method for a comparative quantification of cytokines and receptors involved in the local skin immune reactions. MATERIAL AND METHODS: Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87 bp). The most sensitive PCR-conditions were obtained by optimisation of primer and Mn(OAc)2-concentrations, which led to significant PCR signals (C(T)-value) of less than 36 cycles. A strong correlation between PCR efficiency of the internal control (GAPDH) compared to targets (IL-1beta, IL-10, IL-10r, TNFalpha, IL-7) allowed the use of deltadelta C(T)-method to quantify comparable mRNA levels. RESULTS: Interleukin-1beta (IL-1beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFalpha) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1beta induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MEDjp). Maximal TNFalpha mRNA expression (approx. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA induction reached a maximum of approximately 14-fold 24 h after UV-irradiation of 3 MEDjp. Time- and dose-dependent changes in Interleukin-7 and Interleukin-10 receptor mRNA levels did not occur after UV-irradiation. CONCLUSIONS: Time-distinct gene induction of IL-1beta, TNFalpha and IL-1beta is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.

Adult↗
PubMed2003

[Quantification of tissue perfusion with novel ultrasound methods].

Perfusion describes an important parameter of tissue vitality, e. g. of the myocardium, brain, or the kidney. In malign tumours, perfusion is of particular interest for characterization and prognosis. In addition, new pro- or anti-angiogenic therapies require a functional imaging which is suitable to quantify vascularity. Sonographic methods for the detection of microvascularity, particularly related smaller than those amenable to Doppler ultrasound, are reviewed. The main focus deals with the explanation of contrast-enhanced sonography using replenishment kinetics of microbubbles which provides a comprehensive quantification of tissue perfusion. Alterations of the microvascularity e. g., under anti-angiogenic therapy, can be depicted in experimental studies using this novel approach. Further clinical applications can be the quantification of the perfusion in the myocardium, the brain, or the kidney. New approaches to optimize the theoretical model to describe the replenishment, and novel technical developments are discussed.

Animals↗
PubMed2005

Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase.

Inorganic polyphosphate (iPoP)-linear chains of up to hundreds of phosphate residues-is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP "overplus" were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP "overplus" was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth conditions.

Culture Media↗
PubMed2002

Hyphenated techniques for the characterization and quantification of metallothionein isoforms.

Recent developments in the coupling of highly selective separation techniques such as capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) to element-specific and molecule-specific detectors, such as inductively-coupled plasma mass spectrometry (ICP-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for the characterization and quantification of metallothioneins (MTs) are critically reviewed and discussed. This review gives an update based on the literature over the last five years. The coupling of CE to ICP-MS is especially highlighted. As a result of progress in new interface technologies for CE-ICP-MS, research topics presented in the literature are changing from "the characterization of interfaces by metallothioneins" to the "characterization of metallothioneins by CE-ICP-MS". New applications of CE-ICP-MS to the analysis of MTs in real samples are summarized. The potential of the on-line isotope dilution technique for the quantification of MTs and for the determination of the stoichiometric composition of metalloprotein complexes is discussed. Furthermore, a selection of relevant papers dealing with HPLC-ICP-MS for MT analysis are summarized and compared to those dealing with CE-ICP-MS. In particular, the use of size-exclusion (SE)-HPLC as a preliminary separation step for metallothioneins in real samples prior to further chromatographic or electrophoretic separations is considered. Additionally, the application of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) for the identification of metallothionein isoforms following electrophoretic or chromatographic separation is discussed.

Animals↗
PubMed2002

Quantification of the refrigerants R22 and R134a in mixtures by means of different polymers and reflectometric interference spectroscopy.

The aim of this study was the quantification of vapors of the ozone-depleting refrigerant R22 in the presence of its most important substitute R134a, by the use of the reflectometric interference spectroscopy and polymers as sensitive layers. First, the sorption characteristic of different types of polymers exposed to the vapors of the two analytes was investigated. Then, binary mixtures of the two refrigerants were measured with an array set-up on the basis of six polymer sensors. The measurements were evaluated by the use of neural networks, whereby low limits of detection of 0.45 percentage volume (vol. %)for R22 and 1.45 vol. % for R134a could be established. Additionally, one polar polymer and one microporous polymer were selected for the measurements with a low-cost set-up. The quantification of R22 in the presence of R134a with this low-cost set-up was possible with a limit of detection of 0.44 vol. %, which would enable a fast and economical monitoring at recycling stations.

Air Pollutants↗
PubMed2002

Identification and quantification of metallothionein isoforms and superoxide dismutase in spiked liver extracts using HPLC-ESI-MS offline coupling and HPLC-ICP-MS online coupling.

A two-dimensional chromatographic method for the characterization of metallothionein isoforms (MT) and superoxide dismutase (SOD) in spiked liver extracts was developed for the optimization of extraction procedures from liver samples. Element-specific detection (ICP-MS) and molecule-specific detection (ESI-MS) were applied for maximum species information. A special focus was laid on the quantitative data evaluation (species stoichiometry, calibration with and without matrix, recovery), which is neglected in most MT/SOD publications with hyphenated techniques. Linearity, precision (residual standard deviation of calibration curves <10%), and detection limits (<0.6 mg L(-1) for MT isoforms and 13 mg L(-1) for SOD) prove the suitability of the method for quantification. An alternative quantification is proposed for the extension towards other lesser or even unknown trace element species, especially the native porcine MT and SOD.

Animals↗
PubMed2003

Quantification of DNA in forensic samples.

Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences.

Animals↗
PubMed2004

Quantification of bile acids directly from plasma by MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) has proved to be a useful method for the quantification of bile acids directly from plasma. Six cholic acid derivatives were selected for analysis: taurocholic acid, taurochenodeoxycholic acid, taurolithocholic acid, glycocholic acid, glycochenodeoxycholic acid, and glycolithocholic acid. Solid-phase extraction (SPE) columns were used to preconcentrate and purify the plasma samples. Calibration curves averaged from 3 days were obtained for the bile acids, and then tested for their ability to accurately determine concentrations from one measurement. In summary, a simple, rapid method has been developed for the quantification of bile salts from plasma by MALDI-MS with SPE cleanup.

Bile Acids and Salts↗
PubMed2004

Advances in analytical toxicology: the current role of liquid chromatography-mass spectrometry in drug quantification in blood and oral fluid.

This paper reviews procedures for quantification of drugs in the biosamples blood, plasma, serum, or oral fluid (saliva, etc.) using liquid chromatography coupled with single-stage or tandem mass spectrometry (LC-MS, LC-MS-MS). Such procedures are important prerequisites for competent toxicological judgment and consultation in clinical and forensic toxicology. They cover blood (plasma, serum) analysis of amphetamines and related designer drugs, anesthetics, anticonvulsants, benzodiazepines, opioids, serotonergic drugs, tricyclic antidepressants, neuroleptics, antihistamines, beta-blockers, muscle relaxants, and sulfonylurea-type antidiabetics, and oral fluid analysis of amphetamines and related designer drugs, cocaine, benzoylecgonine, codeine, morphine, enantiomers of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), the nicotine metabolites cotinine and hydroxycotinine, and finally risperidone and its metabolite 9-hydroxyrisperidone. Basic information on the procedures is given in two tables and an example of quantification is illustrated in two figures. The pros and cons of such LC-MS procedures including sample work-up and ion suppression effects are critically discussed.

Chemistry Techniques, Analytical↗
PubMed2005

Detection and quantification of levoglucosan in atmospheric aerosols: a review.

Levoglucosan is a tracer for biomass burning sources in atmospheric aerosol particles. Therefore, much effort has been recently put into developing methods for its quantification. This review describes and compares both established and emerging analytical methods for levoglucosan quantification in ambient aerosol samples, with the special needs of the environmental analytical chemist in mind.

Aerosols↗
PubMed2006

Validated multi-component CZE-UV procedure for the quantification of human hemorphin LVV-H7 in plasma stability studies.

The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin beta, gamma, epsilon or delta chain exhibiting potential pharmaceutical relevance for blood pressure regulation, the treatment of Alzheimer's disease or learning deficiencies. Here we present the development of a multi-component capillary zone electrophoretic method (CZE-UV), allowing the simultaneous quantification of LVV-H7 and four N-terminal degradation products generated in EDTA plasma. Hemorphins in the supernatant of precipitated plasma samples are quantified by external calibration. Validation of the procedure oriented towards international pharmaceutical guidelines and demonstrated excellent linearity (r2 > or = 0.999), good precision (repeatability and reproducibility below 11%), accuracy (-8.4%-4%), ruggedness and an appropriate lower limit of quantification (LLOQ 1.0 microg mL(-1)). This procedure was applied to stability studies of LVV-H7 in human EDTA plasma attended by profiling metabolites using qualitative MALDI-TOF MS analysis. We detected the activity of a soluble plasma form of aminopeptidase M causing successive N-terminal truncation. This is the first time that LVV-H7 degradation as well as its metabolite production have systematically been monitored by a quantitative CZE-UV procedure, underlining the growing importance of such techniques in peptide analysis. In addition, our results give useful hints for future drug development of LVV-H7.

Alzheimer Disease↗
PubMed2007

Validation of a new planar chromatographic method for quantification of the heterocyclic aromatic amines most frequently found in meat.

A new HPTLC method, in which all the HPTLC steps are performed automatically, has been established for quantification of the five most frequently found heterocyclic aromatic amines (HAA: PhIP, MeIQx, 4,8-DiMeIQx, norharmane, and harmane). The method was used for trace analysis (low microg kg(-1) range) of HAA in meat samples and found to be a cost-effective alternative to HPLC, because it enables simultaneous chromatography of up to 20 samples within 30 min. After preconditioning of the HPTLC silica gel layer with ammonia vapour the plate was developed with methanol-chloroform, 1:9 (v/v). At least 4ó separations were obtained and selectivity for the meat matrix was achieved. In repeatability tests relative standard deviations (RSD, n = 14, peak height) were less than or equal to +/-3.3%. In tests of intermediate precision (mean value for 14 tracks on six different plates on six different days) RSDs were better than +/-2% (peak height). Measurement of the reproducibility of migration distances on six different plates resulted in RSDs less than or equal to +/-1.3%. Limits of detection and quantification (S/N 3 and 10, respectively) for the five HAA ranged between 0.4 and 5 ng per band and between 0.8 and 14 ng per band, respectively. With paraffin-n-hexane, 3:7 (v/v), as fluorescence enhancer more sensitive determinations (up to 8.5-fold greater signal intensities) were achieved for PhIP, norharmane, and harmane. In the working range (1:10) polynomial regressions for PhIP, MeIQx, and 4,8-DiMeIQx and linear regressions for norharmane and harmane resulted in RSDs between +/-1.9 and 3.6%. To confirm the absence of potentially coeluting minor HAA, rarely formed in meat, mass spectra were occasionally recorded by online ESI-MS. Robustness tests showed that preconditioning with ammonia was essential for successful separation and that relative humidity had only a minor effect on the chromatography.

Journal Article↗
PubMed2006

Quantification of Lactobacillus in fermented milk by multivariate image analysis with least-squares support-vector machines.

This paper reports an approach for quantification of Lactobacillus in fermented milk, grown in a selective medium (MRS agar), by use of digital colour images of Petri plates easily obtained by use of a flatbed scanner. A one-dimensional data vector was formed to characterize each digital image on the basis of the frequency-distribution curves of the red (R), green (G), and blue (B) colour values, and quantities derived from them, for example lightness (L), relative red (RR), relative green (RG), and relative blue (RB). The frequency distributions of hue, saturation, and intensity (HSI) were also calculated and included in the data vector used to describe each image. Multivariate non-linear modelling using the least-squares support vector machine (LS-SVM) and a linear model based on PLS regression were developed to relate the microbiological count and the frequency vector. Feasibly models were developed using the LS-SVM and errors were below than 10% for Lactobacillus quantification, indicating the proposed approach can be used for automatic counting of colonies.

Animals↗
PubMed2001

Quantification of derivatives of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) migrated from can coatings into tuna by HPLC/fluorescence and MS detection.

A reversed phase high performance liquid chromatographic method combined with fluorescence and mass spectrometric detection in series is presented for the separation and quantification of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) derivatives in extracts from food can coatings, tuna and oil. Fifteen samples of tuna cans bought in four European countries were investigated. Atmospheric pressure chemical ionization mass spectrometry in the positive ion mode (APCI(+)-MS) allowed to tentatively identify BADGE and NOGE related compounds originating from reactions of the glycidyl ethers with bisphenols, phenol, butanol, water and hydrochloric acid. Quantification was based on the external standard method and fluorescence detection. Mass fractions up to 3.7 micrograms/g were found for hydrochlorination products of bisphenol F diglycidyl ether (BFDGE + 2HCl) in tuna. Furthermore, total migration quantities of phenolic ether compounds were estimated. The highest values found were 20 micrograms/g in tuna and 43 micrograms/g in the oil phase.

Animals↗
PubMed2006

Development of stable isotope dilution quantification liquid chromatography-mass spectrometry method for estimation of exposure levels of bisphenol A, 4-tert-octylphenol, 4-nonylphenol, tetrabromobisphenol A, and pentachlorophenol in indoor air.

The potential risks to human health and reproduction of phenolic xenoestrogens, such as bisphenol A (BPA), 4-tert-octylphenol (OP), 4-nonylphenol (NP), tetrabromobisphenol A (TBBPA), and pentachlorophenol (PCP), have not been fully elucidated. It is possible that healthy humans are exposed to phenolic xenoestrogens in their daily lives. It is also possible that the source of human exposure to trace amounts of phenolic xenoestrogens is indoor air. Therefore, we have established an accurate, sensitive, and selective method for determining BPA, OP, NP, TBBPA, and PCP in air samples by using stable isotope dilution techniques using 13C12-BPA, 4-(1-methyl) octylphenol-d5 (m-OP-d5), 13C12-TBBPA, 13C6-PCP, and liquid chromatography-mass spectrometry (LC-MS). Air sampling (71/min, 24 hours), using glass filters (GB-100R, 47 mm) and Empore SDB-XD filters (47 mm/0.5 mm, 3M, Tokyo, Japan) and subsequent extraction by washing were carried out. Then the extracted sample solution was subjected to LC-MS. The percentage recovery of these analytes ranged from 87.0% to 101.9% (SD 0.2% to 4.6%, n=3) with correction using the stable isotope dilution quantification method. The quantification limit (signal-to-noise ratio, S/N>10) in the air samples was 0.1 ng/m3. Our findings suggest that the LC-MS method can detect low levels of phenolic xenoestrogens in air samples and that these trace levels of analytes exist in indoor air.

Air Pollution, Indoor↗
PubMed2005

Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

Bacteria↗
PubMed2002

Molecular cloning, characterization, and quantification of squirrel monkey ( Saimiri sciureus) Th1 and Th2 cytokines.

Primates have long been used as models to study the basic mechanisms of human disease. The squirrel monkey, Saimiri sciureus,is a New World monkey that can be satisfactorily bred in captivity and infected with various human pathogens. The basic immunological parameters of squirrel monkeys, including immunoglobulin subclasses and markers for lymphocyte subsets, have already been characterized. However, immunological reagents and assays specific for the detection and quantification of squirrel monkey cytokines are required to elucidate the immunological and physiopathological changes that occur during experimental infection of these animals with pathogens. We therefore cloned, sequenced, and characterized the cDNAs encoding various squirrel monkey Th1 and Th2 cytokines, including interleukin (IL)-1 beta, IL-2, IL-5, IL-6, IL-10, tumor necrosis factor alpha, and gamma interferon. We found 91.4%-98.1% homology between the nucleotide sequence of the squirrel monkey cytokine genes and published sequences of equivalent human and non-human primate genes. The aligned sequences of cytokines for Saimiriand several New and Old World primates and human are shown, and a phylogenetic analysis of published sequences of selected cytokines in other species and those of non-human primates is given. In addition, we adapted a previously described quantitative, reverse transcriptase, competitive polymerase chain reaction technique to measure the mRNA expression of those cytokines in squirrel monkeys. The primer and competitor plasmids that allowed quantification are described. We showed that the assay is sensitive and reproducible.

Animals↗