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Sample Preparation (sample + preparation)

Distribution by Scientific Domains

Chemistry	66%
Life Sciences	13%
Medical Sciences	8%
Polymers and Materials Science	7%
Earth and Environmental Science	2%
2 Other Domains	4%

Distribution within Chemistry

Mass Spectrometry	37%
General & Introductory Food Science & Technology	4%
Analytical Chemistry	3%
12 Other Subdomains	56%

Terms modified by Sample Preparation

sample preparation method

sample preparation methods

sample preparation procedure

sample preparation protocol

sample preparation step

sample preparation techniques

Selected Abstracts

DEVELOPMENT OF SAMPLE PREPARATION, PRESENTATION PROCEDURE AND SENSORY DESCRIPTIVE ANALYSIS OF GREEN TEA

JOURNAL OF SENSORY STUDIES, Issue 4 2008
SOH MIN LEE
ABSTRACT Although the infusing condition of green tea is critical in determining green tea quality, the green tea industries lack a validated standardized tea preparation procedure. The objectives were (1) to develop an effective sample preparation and presentation procedure to conduct an objective sensory analysis; and (2) to elucidate the effects of green tea types and infusing conditions on the sensory characteristics of green tea. The optimum infusing times for green tea at two temperatures (60 and 80C) were determined using the just-about-right scale evaluated by consumers. Then, a descriptive analysis was conducted. The panelists developed 16 descriptors, and determined the reference samples and the tasting procedure. The optimum infusing time,temperature combinations are approximately 3 min at 60C or 1 min at 80C. The intensity of fermented-like flavor increased, but cut grass and floral flavor decreased with the lower-graded tea leaf. Samples infused at 60C,3 min were sweeter but less bitter than samples at 80C,1 min. PRACTICAL APPLICATIONS The sample preparation method and evaluating conditions developed in this study have been validated using both analytical and consumer studies. The protocols showed to be powerful in discriminating the sensory characteristics between the samples when conducting objective sensory analyses. The sensory lexicons and standards established should be useful to researchers and product developers who are working with flavors of green tea. Additionally, the sample preparation method and evaluation procedure introduced in this study are relatively straightforward, thus, making it possible for the general sensory scientist group to use an effective standardized method when conducting objective sensory analyses of green tea. [source]

,Proteomic Basics , Sample Preparation and Separation': The 1st European Summer School in Kloster Neustift 12,18 August, 2007 Brixen/Bressanone, South Tyrol, Italy

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008
Katrin Marcus Dr.
Abstract Proteomics is rapidly developing into a routine approach for protein analysis in many laboratories. The series of European-wide Summer Schools ,Proteomics Basics' (http://www.proteomic-basics.eu/) aims at teaching of comprehensive knowledge in proteomics research and applied technologies for master and graduate students and postdocs currently moving into the field of proteomic research. In the next 3,years the series will cover the theoretical basis of the fundamental topics in the various areas of proteomic analysis, i.e. sample preparation and handling, mass spectrometry, post-translational modifications and quantitation given by leading experts in the field. This summer school series embodies a unique advantage in comparison with conventional scientific meetings and university curricula: internationally renowned experts will give a detailed perspective view of the fundamentals of their particular proteome research area, something which is usually not encountered at conferences and congresses. Here, we give a report on the first European Summer School ,Sample Preparation and Handling' within the series ,Proteomic Basics' that was held at the monastery in Neustift close to Bressanone/Brixen, Italy from August 12 to 18, 2007. [source]

Influence of Sample Preparation, Staining Procedure and Analysis Conditions on Bull Sperm Head Morphometry using the Morphology Analyser Integrated Visual Optical System

REPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2001
A Boersma
The importance of standardizing the procedures of sample and slide preparation for computer-assisted morphologic analysis has been emphasized in human and veterinary andrology. The purpose of this study was to optimize slide preparation (dilution grade and sperm washing), staining procedures and analysis conditions (colour of light source and objective magnification) for the morphometric analysis of bull spermatozoa using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS). For experiment 1, one ejaculate was collected from one bull and diluted to 200 000,300 000 spermatozoa/,l. Slides were prepared and stained using seven different procedures: rapid Papanicolaou (PAP), rapid Papanicolaou with prolonged staining times (PAP+), Diff-Quik (DIF), haematoxylin (HEM), Farelly (FAR), Spermac (SPER) and the modified GZIN (MGZIN) staining. All slides were analysed using a Hamilton Thorne Morphology Analyser IVOS equipped alternatively with a red, green or blue light source, and a 40× or 100× oil immersion objective. Recognition and digitization errors as well as morphometric parameters were determined. The IVOS was unable to detect DIF-stained spermatozoa. The GZIN and the SPER staining as well as the blue light source led to unsatisfactory results. Among the staining methods examined, the FAR, HEM, PAP+, and PAP staining, preferably in combination with the green light source, and the 40× objective yielded optimal results concerning sperm recognition and digitization. The 100× objective did not allow reliable analysis of the sperm heads because of a frequently appearing digitization error. For experiment 2, three ejaculates were collected from each of three bulls and diluted to five dilution grades (100 000,500 000 spermatozoa/,l). An aliquot of each dilution grade was washed additionally. The percentage of correctly digitized sperm heads decreased with increasing spermatozoal concentration. However, the evaluation speed increased. The range of 200 000,300 000 spermatozoa/,l appeared to be a reasonable compromise for both criteria. Sperm washing failed to further improve the analysis results. Sperm head dimensions were influenced significantly by all variations of the methods in both experiments. In conclusion, using the proposed methods, the IVOS allows precise and reliable morphometric analyses of bull spermatozoa. The consistent application of these procedures may lead to an inter-laboratory standardization and to further establishment of generally accepted morphometric criteria used in human andrology (e.g. World Health Organisation or strict criteria). [source]

THE MOLECULAR FUTURE IN CYTOLOGY

CYTOPATHOLOGY, Issue 2006
M. Salto-Tellez
Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Molecular Diagnosis is, arguably, the fastest growing area of diagnostic medicine. The US market for molecular testing generated $1.3 billion in 2000, which was predicted to increase to about $4.2 billion by 2007.1 We proposed the term Diagnostic Molecular Cytopathology to define the application of molecular diagnosis to cytopathology2. Diagnostic Molecular Cytopathology is essential for the following reasons: (i) Molecular testing is sometimes indispensable to establish an unequivocal diagnosis on cell preparations; (ii) Molecular testing provides extra information on the prognosis or therapy of diseases diagnosed by conventional cytology; (iii) Molecular testing provides genetic information on the inherited nature of diseases that can be directly investigated in cytology samples, by either exfoliation or by fine needle aspiration; (iv) Sometimes the cytopathology sample is the most convenient (or the only available) source of material for molecular testing; (v). Direct molecular interrogation of cells allows for a diagnostic correlation that would otherwise not be possible. Parallel to this direct diagnostic implication, cytopathology is increasing important in the validation of biomarkers for specific diseases, and in therefore of significant importance in the overall translational research strategies. We illustrate its application in some of the main areas of oncology molecular testing, such as molecular fingerprinting of neoplasms,3 lymphoreticular diseases,2 sarcomas4 and lung cancer,5 as well as translational research using diagnostic cytopathology techniques. The next years will see the consolidation of Diagnostic Molecular Cytopathology, a process that will lead to a change of many paradigms. In general, diagnostic pathology departments will have to reorganize molecular testing to pursue a cost-efficient operation. Sample preparation will have to take into account optimal preservation of nuclear acids. The training of technical staff and the level of laboratory quality control and quality assurance would have to follow strict clinical (not research) laboratory parameters. And, most importantly, those pathologists undertaking molecular diagnosis as a discipline would have to develop their professional expertise within the same framework of fellowships and professional credentials that is offered in other sub-specialties. The price to pay if this effort is not undertaken is too important for the future of diagnostic pathology in general. The increasing characterization of molecular biomarkers with diagnostic, prognostic or therapeutic value is making the analysis of tissue and cell samples prior to treatment a more complex exercise. If cytopathologists and histopathologists allow others to take charge of molecular diagnosis, our overall contribution to the diagnostic process will be diminished. We may not become less important, but we may become less relevant. However, those within the discipline of diagnostic pathology who can combine the clinical background of diseases with the morphological, immunocytochemical and molecular diagnostic interpretation will represent bona fide diagnostic specialists. Such ,molecular cytopathologists' would place themselves at the centre of clinical decision-making. Reference:, 1. Liz Fletcher. Roche leads molecular diagnostics charge. Nature Biotechnol 20, 6,7; 2002 2. Salto-Tellez M and Koay ESC. Molecular Diagnostic Cytopathology - Definitions, Scope and Clinical Utility. Cytopathology 2004; 15:252,255 3. Salto-Tellez M, Zhang D, Chiu LL, Wang SC, Nilsson B, and Koay ESC. Immunocytochemistry Versus Molecular Fingerprinting of Metastases. Cytopathology, 2003 Aug; 14(4):186,90. 4. Chiu LL, Koay SCE, Chan NL and Salto-Tellez M. Molecular Cytopathology: Sequencing of the EWS-WT1 Gene Fusion Transcript in the Peritoneal Effusion of a Patient with Desmoplastic Small Round Cell Tumour. Diagnostic Cytopathology, 2003 Dec; 29(6): 341,3. 5. TM Chin, D Anuar, R Soo, M Salto-Tellez, WQ Li, B Ahmad, SC Lee, BC Goh, K Kawakami, A Segal, B Iacopetta, R Soong. Sensitive and Cost-Effective deptection of epidermal growth factor Receptor Mutations in Small Biopsies by denaturing High Performance Liquid Chromatography. (In press). [source]

Historical review of sample preparation for chromatographic bioanalysis: pros and cons

DRUG DEVELOPMENT RESEARCH, Issue 3 2007
Min S. Chang
Abstract Sample preparation is a major task in a regulated bioanalytical laboratory. The sample preparation procedure significantly impacts assay throughput, data quality, analysis cost, and employee satisfaction. Therefore, selecting and optimizing an appropriate sample preparation method is essential for successful method development. Because of our recent expertise, this article is focused on sample preparation for high-performance liquid chromatography with mass spectrometric detection. Liquid chromatography with mass spectrometric detection (LC-MS) is the most common detection technique for small molecules used in regulated bioanalytical laboratories. The sample preparation technologies discussed are pre-extraction and post-extraction sample processing, protein precipitation (PPT), liquid,liquid extraction (LLE), offline solid-phase extraction (SPE), and online solid-phase extraction. Since all these techniques were in use for more than two decades, numerous applications and variations exist for each technique. We will not attempt to categorize each variation. Rather, the development history, a brief theoretical background, and selected references are presented. The strengths and the limitations of each method are discussed, including the throughput improvement potential. If available, illustrations from presentations at various meetings by our laboratory are used to clarify our opinion. Drug Dev Res 68:107,133, 2007. ©2007 Wiley-Liss, Inc. [source]

Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control.

DRUG TESTING AND ANALYSIS, Issue 8 2009

Abstract Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 µg mL,1 (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1,20 µg mL,1. The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Competitive immunoassay by capillary electrophoresis with laser-induced fluorescence for the trace detection of chloramphenicol in animal-derived foods

ELECTROPHORESIS, Issue 16 2008
Can Zhang
Abstract A competitive immunoassay using CE with an LIF detector was developed for the detection of chloramphenicol (CAP). The method was based on the competitive reactions between fluorescently labeled CAP hapten and free CAP, with a limited amount of anti-CAP antibody. The poly(N -isopropylacrylamide) (pNIPA) hydrogel was added in the separation buffer as a dynamic modifier to reduce adsorption and enhance reproducibility. The linear range and LOD for CAP were 0.008,5,,g/L and 0.0016,,g/L, respectively. An ELISA using the same immuno-reagents was also developed for the analysis of CAP, with an LOD of 0.03,,g/L. The sensitivity of this CE immunoassay (CEIA)-LIF was almost 20 times greater than that of the ELISA. Using CEIA-LIF, equilibrium was reached in 15,min and the analytical results were obtained within 5,min by CE separation. Sample preparation for CEIA-LIF was not time-consuming and the matrix effect was easy to remove. An LOD of 0.1,,g/kg CAP in food matrices was easily achieved. This method is thus proposed as a fast and sensitive means of detecting trace amounts of CAP residues in animal-derived foods. [source]

Determination of ribavirin in human serum and plasma by capillary electrophoresis

ELECTROPHORESIS, Issue 10-11 2004
Michael C. Breadmore
Abstract The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 ,L sample and reconstitution of the dried extract into 100 ,L of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 ,g/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-,-2b for a hepatitis C virus infection. [source]

Neutron Activation Analysis, Atomic Absorption and X-Ray Fluorescence Spectrometry Review for 2006,2007

GEOSTANDARDS & GEOANALYTICAL RESEARCH, Issue 4 2008
L. Paul Bédard
These mature analytical techniques do not show any change in publication level from the previous two years and AAS remains dominant in terms of the number of publications. The last two years have seen fewer technical improvements than in the previous review period. Some interesting papers dealing with uncertainty and quality assurance in INAA were published during 2006,2007. It is suggested that photon activation should be reconsidered because the source of electron accelerators has recently improved. A technique to preconcentrate Se for INAA determination has also been proposed. In the case of AAS, papers on analyte preconcentration continue to be more abundant than those relating to instrumental modification. Sample preparation for AAS is also active and ultrasound-assisted leaching shows some promising applications. There were an unusual number of reviews concerned with AAS and those important to geological samples are cited here. A technique to preconcentrate Cr in water is presented and a new device to determine As and Se is showing some potential uses. Confocal X-ray mapping continues to show interesting developments. One group developed a technique to perform XRF inside an oyster and an interesting application of ,-XRF mapping of sediments is presented. Determination of platinum-group elements (at ,g g1 concentrations) can be carried out very quickly with an improved XRF technique. [source]

Challenges and Progress in High-Throughput Screening of Polymer Mechanical Properties by Indentation

ADVANCED MATERIALS, Issue 35 2009
Johannes M. Kranenburg
Abstract Depth-sensing or instrumented indentation is an experimental characterization approach well-suited for high-throughput investigation of mechanical properties of polymeric materials. This is due to both the precision of force and displacement, and to the small material volumes required for quantitative analysis. Recently, considerable progress in the throughput (number of distinct material samples analyzed per unit time) of indentation experiments has been achieved, particularly for studies of elastic properties. Future challenges include improving the agreement between various macroscopic properties (elastic modulus, creep compliance, loss tangent, onset of nonlinear elasticity, energy dissipation, etc.) and their counterpart properties obtained by indentation. Sample preparation constitutes a major factor for both the accuracy of the results and the speed and efficiency of experimental throughput. It is important to appreciate how this processing step may influence the mechanical properties, in particular the onset of nonlinear elastic or plastic deformation, and how the processing may affect the agreement between the indentation results and their macroscopic analogues. [source]

Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

INSECT SCIENCE, Issue 6 2008
Li-Zhen Chen
Abstract Brush border membrane vesicles (BBMV) isolated from insect midguts have been widely used to study Cry1A binding proteins. Sample preparation is important in two-dimensional electrophoresis (2-DE), so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2-DE analysis. [source]

Rietveld quantitative amorphous content analysis

JOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2001
A. G. De La Torre
A procedure for Rietveld quantitative amorphous content analysis (RQACA) is outlined, in which the effects of systematic errors in the powder patterns are studied. The method derives the amorphous content from the small overestimation of an internal crystalline standard in a Rietveld refinement of an appropriate mixture. Of several standards studied, Al2O3 gave the best results. The statistical analysis of standard mixtures with a known amount of amorphous content indicated that this is a precise and accurate tool. It enables the measurement of the amorphous content with an accuracy close to 1%. Sample preparation and Rietveld analysis need to be optimized in order to minimize the systematic errors. The analysis of samples with phases displaying strong preferred orientation effects gives very high errors in the amorphous content. Samples with different absorption coefficients have also been studied in order to evaluate the importance of microabsorption. This plays an important role but it can be adequately corrected if the absorption coefficients of the standard and the sample are not very different. RQACA has been applied to tricalcium silicate, C3S, which is the main component of Portland cement. The average amorphous content of C3S, after microabsorption correction using two standards of higher and lower absorption coefficients, was found to be 19%. [source]

Use of the 1-mm micro-probe for metabolic analysis on small volume biological samples

JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009
Natalie J. Serkova
Abstract Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and 1H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 ,l: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 = 106: chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count. [source]

Determination of clavulanic acid in calf plasma by liquid chromatography tandem mass spectrometry

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2006
Tim Reyns
Abstract A method for the quantification of clavulanic acid in calf plasma using high-performance liquid chromatography combined with electrospray ionization (ESI) mass spectrometry, operating in the negative ionization mode (LC-MS/MS), is presented. Sample preparation includes a simple and fast deproteinization with acetonitrile and a back-extraction of the acetonitrile with dichloromethane. Chromatography is performed on a reversed-phase PLRP-S polymeric column using 0.05% formic acid in water and acetonitrile. The limit of quantification is 25 ng/ml, which is lower than other published methods using ultraviolet (UV), fluorimetric or mass spectrometric detection. The limit of detection is calculated to be 3.5 ng/ml. The stability of clavulanic acid was demonstrated according to The Guidelines of Bioanalytical Method Validation of The Food and Drug Administration (FDA): freeze and thaw stability, short-term stability, long-term stability, stock solution stability and postpreparative stability. The method is used in a pharmacokinetic and bioequivalence study of amoxycillin/clavulanic acid formulations in calves. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Hollow-fibre supported liquid membrane extraction for determination of fluoxetine and norfluoxetine concentration at ultra trace level in sewage samples

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 15 2007
Saioa Zorita
Abstract In this study, a method was developed for determining the concentration of the pharmaceutical fluoxetine and its metabolite, norfluoxetine, in sewage water samples. Sample preparation was performed by hollow-fibre supported liquid membrane (HF-SLM) extraction with final analysis using liquid chromatography with UV detection. Several parameters were studied including type of organic solvent, sample and acceptor pH, and salt and humic acid content. The optimised method allowed determination of the analyte at the ng/L level in sewage water. A linear plot gave a correlation coefficient better than 0.991 for both analytes and resulted in limits of detection in sewage water of 11 and 12 ng/L, for fluoxetine and norfluoxetine, respectively. The enrichment factor was over 1700 for both analytes in sewage water. The repeatability and reproducibility were better than 8% and 17%, respectively. The developed methodology was used to study daily variations of fluoxetine and norfluoxetine in municipal sewage streams. Norfluoxetine has been detected for the first time in sewage water and a preliminary analysis gave average concentrations of 150 and 225 ng/L for norfluoxetine and fluoxetine, respectively. [source]

Continuous mode of operation for large volume dosing in analytical carrier ampholyte-free isoelectric focusing of proteins applied to off-line detection of fractions

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 11 2006
Jana Budilová
Abstract Mass spectrometry is being increasingly used for analysis of proteome complex samples. Sample preparation is often necessary to remove matrix interferences and to concentrate analytes prior to MS measurement. A useful method for this purpose is Carrier Ampholyte Free-Isoelectric Focusing (CAF-IEF). In this paper CAF-IEF of ampholytes was performed on a commercial apparatus EA101 (Villa Labeco, Slovakia) equipped with a specially made column for samples of large volume (up to 0.5 mL). A new continuous mode without voltage interruption or electrolyte replacement was developed. In this mode, a low molecular mass pI marker (PIM 7.4) and low concentrations of myoglobin and insulin (16 mg/L), respectively, were concentrated, and then 5-,L fractions collected for off-line analyses. The total time of focusing was 66 minutes. The concentration of PIM 7.4 in the fractions was increased up to 75 times (determined by UV-VIS spectrometry). The concentration in the fractions was increased up to 30 times for myoglobin and 10 times for insulin. [source]

Direct Preparation of Nanoscale Thin Films of Poly(vinylidene fluoride) Containing , -Crystalline Phase by Heat-Controlled Spin Coating

MACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 24 2008
Subramaniyan Ramasundaram
Abstract Nanoscale thin films of PVDF containing the , -crystalline phase were directly prepared by heat-controlled spin coating without the influence of external stimuli either in the form of additives or post-treatments. Sample preparation was carried out at different temperatures, ranging from 10 to 70,°C. At elevated temperatures (40, 50, 60 and 70,°C), PVDF was crystallized into the , -phase, while at near-ambient conditions (20 and 30,°C) it was crystallized into the , -phase. Some samples exhibited a phase-segregated morphology, with varying particle sizes depending on the preparation temperature. The ferroelectric nature of a typical sample, prepared at 40,°C, was visualized by piezoresponse imaging studies. [source]

Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysis

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005
Lei Huang
Abstract Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95,99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein,protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics. [source]

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2009
Alejandro M. Cohen
This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in-gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A characteristic band around a molecular mass of 185,kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI-MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one-step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high-performance liquid chromatography (HPLC) coupled in-line to an electrospray quadrupole-hexapole-quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0,,,1020.4 and 750.0,,,1205.4) and (754.8,,,1028.6 and 754.8,,,1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Liquid chromatography/electrospray ionization tandem mass spectrometry validated method for the simultaneous quantification of sibutramine and its primary and secondary amine metabolites in human plasma and its application to a bioequivalence study

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2006
Deepak S. Jain
A high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the estimation of sibutramine and its two metabolites (M1 and M2). The extraction of sibutramine, its metabolites and imipramine (internal standard (IS)) from the plasma involved treatment with phosphoric acid followed by solid-phase extraction (SPE) using a hydrophilic-lipophilic balanced HLB cartridge. The SPE eluate without drying and reconstitution was analyzed by LC/MS/MS, equipped with a with turbo ion spray (TIS) source, operating in the positive and multiple reaction monitoring (MRM) acquisition mode. Sample preparation by this method yielded extremely clean extracts with quantitative and consistent mean recoveries; 95.12% for sibutramine, 92.74% for M1, 95.97% for M2 and 96.60% for the IS. The total chromatographic run time was 3.0,min with retention times of 2.51, 2.13, 2.09,min for sibutramine, M1, M2 and imipramine, respectively. The developed method was validated in human plasma matrix, with a sensitivity of 0.1,ng/mL (coefficient of variance (CV), 2.07%) for sibutramine, 0.1,ng/mL (CV, 3.59%) for M1 and 0.2,ng/mL (CV, 4.93%) for M2. Validation of the method for its accuracy, precision, recovery, matrix effect and stability was carried out especially with regard to real subject sample analysis. The response was linear over the dynamic range 0.1 to 8.0,ng/mL for sibutramine and M1, and 0.2 to 16.0,ng/mL for M2 with correlation coefficients of r,,,0.9959 (sibutramine), 0.9935 (M1) and 0.9943 (M2). The method was successfully applied for bioequivalence studies in 40 human subjects with 15,mg capsule formulations. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005
Alexandru C. Lazar
Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2005
Anne K. Callesen
Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and , -cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000,12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Semi-automated quantification of ivermectin in rat and human plasma using protein precipitation and filtration with liquid chromatography/tandem mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2004
Tony Pereira
Ivermectin is a parasiticide commonly used in humans and livestock. It is currently under development for the treatment of pediculosis of humans (head lice) that does not respond to established treatments. A liquid chromatography/turbo ion spray tandem mass spectrometry (LC/TIS-MS/MS) method for the determination of ivermectin in rat and human plasma has been developed that uses emamectin [4,-epi-(methylamino)-4,-deoxyavermectin] as the internal standard. Sample preparation involved protein precipitation and filtration of fortified plasma in the 96-well format. Chromatographic separation was accomplished using fast gradient conditions on a C8 stationary phase. The analytes were detected with the mass spectrometer operated in the positive ion, multiple reaction monitoring mode. The method exhibited good intra- and interday accuracy and precision, and was linear over a dynamic range of 1,2000,ng/mL. In rat plasma, intraday accuracy ranged between 84,93% for the low quality control (QC) sample (1.5 ng/mL), and between 91,109% for the remaining QCs. Intraday precision ranged between 4.9,15% for the low QC, and 0.8,6.3% for the remaining QCs. Interday accuracy ranged between 88,107%, and precision between 4.1,11%. Similar data was obtained using human plasma. An investigation of matrix effects indicated that the ionization efficiency of ivermectin was favored by the presence of an ammonium ion in an aqueous environment. The implications of this observation toward assay sensitivity are discussed. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2003
Aldo Laganà
A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10,ng/g for fusarenon X and 1.5,ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives. Copyright © 2003 John Wiley & Sons, Ltd. [source]

HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies

BIOMEDICAL CHROMATOGRAPHY, Issue 6 2009
Soo Kyung Bae
Abstract A simple and rapid HPLC method using fluorescence detection was developed for determination of irbesartan in human plasma. Sample preparation was accomplished through a simple deproteinization procedure with 0.4 mL of acetonitrile containing 800 ng/mL of losartan (internal standard), and to a 0.1 mL plasma sample. Chromatographic separation was performed on a Zorbax Xclipse XDB C18 column (150 × 4.6 mm, i.d., 5 µm) at 40°C. An isocratic mobile phase, acetonitrile:0.1% formic acid (37:63, v/v), was run at a flow-rate of 1.0 mL/min, and the column eluent was monitored using a fluorescence detector set at excitation and emission wavelengths of 250 and 370 nm, respectively. The retention times of irbesartan and losartan were 4.4 and 5.9 min, respectively. This assay was linear over a concentration range of 10,5000 ng/mL with a lower limit of quantification of 10 ng/mL. The coefficient of variation for this assay precision was less than 8.48%, and the accuracy exceeded 94.4%. The mean relative recoveries of irbesartan and losartan were 98.4 and 99.1%, respectively. This method was successfully applied for pharmacokinetic study after oral administration of irbesartan (300 mg) to 23 Korean healthy male volunteers. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Liquid chromatographic/mass spectrometry assay of bromotetrandrine in rat plasma and its application to pharmacokinetic study

BIOMEDICAL CHROMATOGRAPHY, Issue 6 2009
Naining Song
Abstract A rapid and sensitive liquid chromatography,tandem mass spectrometric method (LC-MS/MS) for the determination of bromotetrandrine in rat plasma has been developed and applied to pharmacokinetic study in Sprague,Dawley (SD) rats after a single oral administration. Sample preparation involves a liquid,liquid extraction with n -hexane,dichlormethane (65:35, containing 1% 2-propanol isopropyl alcohol, v/v). Bromotetrandrine and brodimoprim (internal standard, IS) were well separated by LC with a Dikma C18 column using methanol,ammonium formate aqueous solution (20 mm) containing 0.5% formic acid (60:40, v/v) as mobile phase. Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 703.0 , 461.0 and m/z 339.0 , 281.0 for bromotetrandrine and IS, respectively. The present method exhibited good linearity over the concentration range of 20,5000 ng/mL for bromotetrandrine in rat plasma with a lower limit of quantification of 20 ng/mL. The intra- and inter-day precisions were 2.8,7.5% and 3.2,8.1%, and the intra- and inter-day accuracy ranged from ,4.8 to 8.2% and ,5.6 to 6.2%, respectively. The method was successfully applied to a pharmacokinetic study after a single oral administration to SD rats with bromotetrandrine of 50 mg/kg. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Simultaneous determination of 18, - and 18, -glycyrrhetic acid in human plasma by LC-ESI-MS and its application to pharmacokinetics

BIOMEDICAL CHROMATOGRAPHY, Issue 1 2009
Qiaogen Zou
Abstract A highly sensitive and selective LC-ESI-MS was developed, validated for the simultaneous determination of 18, -glycyrrhetic acid (, -GA) and 18,-glycyrrhetic acid (, -GA) for pharmacokinetic studies in healthy subjects. Sample preparation was performed by liquid,liquid extraction with ethyl acetate and the separations were achieved using a C18 column with the mobile phase composed of 10 mmol/L ammonium acetate solution,methanol,acetonitrile (40:36:24, v/v/v) at a flow rate of 1 mL/min. The internal standard was honokiol and the epimers were quantified using a single quadrupole mass spectrometer employing ESI in the negative ion mode. The separation factor, ,, was 1.512 for , - and , -GA. The standard curves were linear for both epimers with coefficients of determination (r , 0.9998) over the concentration range of 1,150 ng/mL. The precision and accuracy were , 4.33 and , 6.57%, respectively. The mean plasma extraction recoveries were 82.23 ± 1.91 and 84.29 ± 2.09% for , -GA and , -GA, respectively. The assay was successfully applied to evaluation of the pharmacokinetic properties of , -GA and , -GA from 19 volunteers who had received oral administration of diammonium glycyrrhizinate capsules. The initial data suggest that glycyrrhizin metabolizes to glycyrrhetic acid fairly slowly and the elimination of , -GA is slower than that of , -GA. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Improved RP-HPLC determination of quinine in plasma and whole blood stored on filter paper

BIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2000
J.A. Kolawole
Abstract Analysis of quinine in plasma and whole blood samples dried on filter paper is described. Sample preparation involves liquid extraction of plasma and whole blood from the filter paper and subsequent solid-phase extraction using C8 Bond Elut cartridges. A reverse-phase liquid chromatography system with UV detection and fluorescence detection was used. The analytical characteristics of the method are reported, with a quantification limit of 0.1 µg mL,1 and within an assay coefficient of variation of 5.6,8.4% in plasma and 6.5,12% in whole blood. Representative chromatograms are shown as a function of time for samples from human subjects after ingestion of a single 400-mg dose of quinine sulphate. Quinidine, dihydroquinine and metabolites are well separated from quinine with a resolution of above 1 (Rs>1). Copyright © 2000 John Wiley & Sons, Ltd. [source]

Simultaneous determination of avermectins in bovine tissues by LC-MS/MS

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 21 2009
Koichi Inoue
Abstract Analytical method for the simultaneous quantification of avermectins (AVMs), abamectin B1a, abamectin 8,9-Z isomer B1a, emamectine benzoate B1a, emamectine benzoate 8,9-Z isomer B1a, ivermectin, eprinomectin B1a, doramectin and moxidectin in bovine tissues (muscle, liver and fat) was developed by LC-MS/MS in electrospray positive ion mode. The separation was achieved on a short TSK-GEL ODS 100V column with the mobile phase consisting of acetonitrile and aquatic 0.1,mM ammonium formate containing 0.1% formic acid v/v at a flow rate of 0.2,mL/min with gradient elution. Liquid,liquid extraction with isooctane was used for the sample extraction/preparation of analytes in bovine samples. The linearity of the calibration curves was excellent in matrix-matched standards, and yielded the coefficients (r2=0.997,0.999, range from LOQ to 500, 1000 or 5000,ng/g) of determination of the target analytes. Recoveries were in the range of 87.9,99.8% with associated precision values (within-day: 1.5,7.4%, n=6, and between-day: 1.5,8.4% for 3 days) for repeatability and reproducibility. LC-MS/MS method has been proven to be highly efficient and suitable for the simultaneous determinations of eight AVMs in bovine tissue samples. [source]