Samples Of Biological Origin Research Articles

Iron Speciation in Animal Tissues Using AC Magnetic Susceptibility Measurements: Quantification of Magnetic Nanoparticles, Ferritin, and Other Iron-Containing Species.

The simultaneous detection and quantification of several iron-containing species in biological matrices is a challenging issue. Especially in the frame of studies using magnetic nanoparticles for biomedical applications, no gold-standard technique has been described yet and combinations of different techniques are generally used. In this work, AC magnetic susceptibility measurements are used to analyze different organs from an animal model that received a single intratumor administration of magnetic nanoparticles. The protocol used for the quantification of iron associated with the magnetic nanoparticles is carefully described, including the description of the preparation of several calibration standard samples of nanoparticle suspensions with different degrees of dipolar interactions. The details for the quantitative analysis of other endogenous iron-containing species such as ferritin or hemoglobin are also described. Among the advantages of this technique are that tissue sample preparation is minimal and that large amounts of tissue can be characterized each time (up to hundreds of milligrams). In addition, the very high specificity of the magnetic measurements allows for tracking of the nanoparticle transformations. Furthermore, the high sensitivity of the instrumentation results in very low limits of detection for some of the iron-containing species. Therefore, the presented technique is an extremely valuable tool to track iron oxide magnetic nanoparticles in samples of biological origin.

Problematic Issues on the Organization of Selecting Person’s Biological Samples for Molecular and Genetic Examination

The article is focused on the procedure of sampling as a criminal procedural action necessary for the commissioning and carrying out molecular genetic expert examination in the divisions of the Expert Service of the Ministry of Internal Affairs, namely the establishment and comparison of genetic traits of living and dead persons and unidentified corpses within criminal proceedings and civil cases for establishing or refutation of the fact of paternity. The purpose of the work is to provide information about human biological material that can be used as samples for molecular genetic research of living and dead people and unidentified corpses; analysis of errors during sampling of biological material of individuals for each type of samples and determining the methods of storage and transportation of research objects depending on their condition. The author has studied the legal aspects of the procedure of selecting the samples of biological origin according to the rules of identity examination and has indicated the list of documents required for sampling relatives of missing persons for the placement in the central database of human genetic traits. The author has specified the basic requirements for sampling, such as their indisputable belonging to a certain person and reliability of their selection in the minimum quantity necessary for the research. The information that should be indicated on the packaging of biological material has been described. The types of biological material that can be provided for the research as living specimens have been separately studied, namely: nails, blood, buccal epithelium, saliva, which can be provided on a piece of sterile gauze, FTA Cards, cotton, viscose or flock sticks or probe swabs. The author has also defined the persons who have the right to carry out selection from living persons, conditions of selection, drying of samples, possibilities of their long-term storage and conditions of transportation to divisions of the Expert Service of the Ministry of Internal Affairs of Ukraine. Errors in selecting the packaging of biological material (catheters with heparin filling, Ames medium, polymer containers) have been analyzed. Blood, bone remains, teeth, nails and histological specimens have been studied as types of biological material taken from dead persons and unidentified corpses depending on the condition of the remains, environmental conditions and prescription of death coming.

An Improved Methodology for Determination of Fluorine in Biological Samples Using High-Resolution Molecular Absorption Spectrometry via Gallium Fluorine Formation in a Graphite Furnace

Nowadays growing attention is paid to the control of fluorine content in samples of biological origin as it is present in the form of various biologically active organic compounds. Due to the chemically-rich matrix of biological tissues, the determination of fluorine becomes a very difficult task. Furthermore, a required complex sample preparation procedure makes the determination of the low contents of F by ion chromatography UV-Vis or ion-selective electrodes not possible. High-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) seems to be the best option for this purpose due to its high robustness to matrix interferences, especially in the presence of carefully selected modifiers. In this work the possibility of quantitative F determination in water and animal tissues was examined by measuring the molecular absorption of gallium monofluoride (GaF) at 211.248 nm with the use of a commercially available HR-CS GF MAS system. Experimental conditions for the sensitive and precise determination of fluorine were optimized, including the time/temperature program as well as addition of gallium and modifier mixture in combined mode. Under these conditions the fluoride present in the sample was stabilized up to 600 °C, and the optimum vaporization temperature for GaF was 1540 °C. Palladium and zirconium deposited onto the graphite surface served as solid modifiers; sodium acetate and ruthenium modifiers were added directly to the sample. The limit of detection and the characteristic mass of the method were 0.43 μg/L and 8.7 pg, respectively. The proposed procedure was validated by the use of certified reference materials (CRMs) of lake water and animal tissue; the acceptable recovery was obtained, proving that it can be applied for samples with a similar matrix.

Sample preparation for Raman microspectroscopy

Abstract Raman spectroscopy and its variants allow for the investigation of a wide range of biological and biomedical samples, i. e. tissue sections, single cells and small molecules. The obtained information is on a molecular level. By making use of databases and chemometrical approaches, the chemical composition of complex samples can also be defined. The measurement procedure is straight forward, however most often sample preparation protocols must be implemented. While pure samples, such as high purity powders or highly concentrated chemicals in aqueous solutions, can be directly measured without any prior sample purification step, samples of biological origin, such as tissue sections, pathogens in suspension or biofluids, food and beverages often require pre-processing steps prior to Raman measurements. In this book chapter, different strategies for handling and processing various sample matrices for a subsequent Raman microspectroscopic analysis were introduced illustrating the high potential of this promising technique for life science and medical applications. The presented methods range from standalone techniques, such as filtration, centrifugation or immunocapture to innovative platform approaches which will be exemplary addressed. Therefore, the reader will be introduced to methods that will simplify the complexity of the matrix in which the targeted molecular species are present allowing direct Raman measurements with bench top or portable setups.

Verification of the Major Metabolic Oxidation Path for the Naphthoyl Group in Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells (CRTh2) Antagonist 2-(2-(1-Naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic Acid (Setipiprant/ACT-129968).

Various racemic and enantioenriched (trans)-X,Y-dihydroxy-X,Y-dihydronaphthoyl analogues as well as X-hydroxy-naphthoyl analogues of CRTh2 antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (1, Setipiprant/ACT-129968) were synthesized in order to gain insight into regio- and enantioselectivity of the metabolic oxidation of 1 and to verify the structures of four metabolites that were proposed earlier in a clinical ADME study. Analytical data of the synthetic standards were compared with data from samples of biological origin. The two major metabolites M7 and M9 were unambiguously verified as 2-(2-((trans)-3,4-dihydroxy-3,4-dihydronaphthalene-1-carbonyl)- and 2-(2-((trans)-5,6-dihydroxy-5,6-dihydronaphthalene-1-carbonyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid, respectively, each composed of two enantiomers with 68% and 44% ee in favor of (+)-(3S,4S)-M7 and (+)-(5S,6S)-M9, respectively. Likewise, minor metabolites M3 and M13 were identified as 2-(8-fluoro-2-(5-hydroxy-1-naphthoyl)- and 2-(8-fluoro-2-(4-hydroxy-1-naphthoyl)-1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indol-5-yl)acetic acid, respectively.

Selective removal of phosphate for analysis of organic acids in complex samples

Accurate quantitation of compounds in samples of biological origin is often hampered by matrix interferences one of which occurs in GC-MS analysis from the presence of highly abundant phosphate. Consequently, high concentrations of phosphate need to be removed before sample analysis. Within this context, we screened 17 anion exchange solid-phase extraction (SPE) materials for selective phosphate removal using different protocols to meet the challenge of simultaneous recovery of six common organic acids in aqueous samples prior to derivatization for GC-MS analysis. Up to 75% recovery was achieved for the most organic acids, only the low pKa tartaric and citric acids were badly recovered. Compared to the traditional approach of phosphate removal by precipitation, SPE had a broader compatibility with common detection methods and performed more selectively among the organic acids under investigation. Based on the results of this study, it is recommended that phosphate removal strategies during the analysis of biologically relevant small molecular weight organic acids consider the respective pKa of the anticipated analytes and the detection method of choice.

Analysis of Chloramphenicol in Biological Samples by SPE-HPLC

: Chloramphenicol (CAP) is a widely used antibiotic for bacterial diseases for animals and in typhoid fever in human beings. SPE-HPLC method is described for the detection of chloramphenicol from human blood plasma samples. Frusemide (FRU) was taken as an internal standard to determine the percentage recovery of chloramphenicol in blood sample. Their standard solutions were prepared in methanol having the concentration of 1.0 mg/mL. The extraction of both the drugs was carried out by SPE using methanol at a flow rate of 0.1mL/min. Further, the analysis was done by HPLC having monolithic, chromolith performance RP-18e, 100-4.6 (100 × 4.6 mm) column. Phosphate buffer (100 mM, pH 2.5)-acetonitrile (75:25, v/v) was selected as a mobile phase at a flow rate of 1.5 mL/min. Detection of the drugs were carried out at 270 nm. The percentage recoveries of CAP and FRU from plasma samples were 92.0 and 90.0, respectively. The detection limit of the developed method for CAP and FRU were 0.8 µg/mL and 1.0 µg/mL, respectively. The result of separation factor (α) and resolution factor (Rs) of CAP with respect to FRU in plasma samples were 2.31 and 16.25, respectively. The developed method may be used for quantification and detection of CAP from samples of biological origin.

Determination of the natural abundance δ15 N of taurine by gas chromatography-isotope ratio measurement mass spectrometry

The measurement of the nitrogen isotope ratio of taurine (2-aminoethanesulphonic acid) in biological samples has a large number of potential applications. Taurine is a small water-soluble molecule which is notoriously difficult to analyze due to its polarity and functionality. A method is described which allows the determination of the natural abundance δ(15)N values of taurine and structural analogues, such as 3-amino-1-propanesulphonic acid (APSA), by isotope ratio mass spectrometry interfaced to gas chromatography (GC-irm-MS). The one-step protocol exploits the simultaneous derivatization of both functionalities of these aminosulphonic acids by reaction with triethylorthoacetate (TEOA). Conditions have been established which ensure quantitative reaction thus avoiding any nitrogen isotope fractionation during derivatization and workup. The differences in the δ(15)N values of derivatized and non-derivatized taurine and APSA all fall within the working range of 0.4‰ (-0.02 to 0.39‰). When applied to four sources of taurine with various δ(15)N values, the method achieved excellent reproducibility and accuracy. The optimized method enables the determination of the natural abundance δ(15)N values of taurine over the concentration range 1.5-7.84 µmol.mL(-1) in samples of biological origin.

Warping and alignment technologies for inter-sample feature correspondence in 1D H-NMR, chromatography-, and capillary electrophoresis-mass spectrometry data

Comprehensive analysis is an emerging and interesting mode of analyzing biological samples. The hypothesis is that by measuring the abundance of as many analytes as possible in samples of biological origin, new knowledge about biological regulation and pathways and can be gained. This hypothesis is one of the cornerstones of the -omics sciences. The comprehensive measurements are usually performed by H-NMR or hyphenated mass spectrometry of almost intact biofluids and tissues. The data generated from these measurements are very complex, containing thousands of analyte signals, necessitating automatic information extraction. The success of this information extraction is dependent on many steps and a most crucial step is solving the inter-sample correspondence problem, i.e. that analytes from different samples are unsynchronized in the measured data. This problem hampers the data-analysis pipeline by introducing errors in the resulting extracted abundance data. The remedy is usually tedious manual inspection for analyte synchronization. In this paper, we address automated methods for addressing the inter-sample correspondence problem when analyzing data from comprehensive measurements, i.e. synchronization methods. We here present a review based on methods taxonomy, complexity, and an overview of current solution technologies available.

Characterization of Chitin and Its Hydrolysis to GlcNAc and GlcN

Proton NMR spectra of chitin dissolved in concentrated and deuterated hydrochloric acid (DCl) were found to be a simple and powerful method for identifying chitin from samples of biological origin. During the first hour after dissolving chitin in concentrated DCl (25 degrees C), insignificant de-N-acetylation occurred, meaning that the fraction of acetylated units (FA) of chitin could be determined. FA of demineralized shrimp shell samples treated with 1 M NaOH at 95 degrees C for 1-24 h were determined and were found to decrease linearly with time from 0.96 to 0.91 during the treatment with NaOH. Extrapolation to zero time suggested that chitin from shrimp shells has a FA of 0.96, that is, contains a small but significant fraction of de-N-acetylated units. Proton NMR spectra of chitin ( FA = 0.96) dissolved in concentrated DCl were obtained as a function of time until the samples were almost quantitatively hydrolyzed to the monomer glucosamine (GlcN). The initial phase of the reaction involves mainly depolymerization of the chitin chains, resulting in that almost 90% (molar fraction) of the chitin is converted to the monomer N-acetyl-glucosamine (GlcNAc).Thus, effective conversion of chitin to GlcNAc in concentrated acid is reported for the first time. GlcNAc is then further de-N-acetylated to GlcN. A new theoretical model was developed to simulate the experimental data of the kinetics of hydrolysis of chitin in concentrated acid. The model uses three different rate constants; two for the hydrolysis of the glycosidic linkages following an N-acetylated or a de-N-acetylated sugar unit and one for the de-N-acetylation reaction. The three rate constants were estimated by fitting model data to experimental results. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher as compared to the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. Two chitin samples with different F A values (0.96 and 0.70) were incubated in concentrated DCl until the samples were converted to the maximum yield of GlcNAc and the oligomer composition analyzed, showing that the maximum yield of GlcNAc was much higher when prepared from the chitin with the highest F A value.

Extraction, separation, and detection methods for phenolic acids and flavonoids

The impetus for developing analytical methods for phenolic compounds in natural products has proved to be multifaceted. Hundreds of publications on the analysis of this category of compounds have appeared over the past two decades. Traditional and more advanced techniques have come to prominence for sample preparation, separation, detection, and identification. This review provides an updated and extensive overview of methods and their applications in natural product matrices and samples of biological origin. In addition, it critically appraises recent developments and trends, and provides selected representative bibliographic examples.

Metabonomics in Diabetes Research

Metabonomics has been defined as "quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification" and can provide information on disease processes, drug toxicity, and gene function. In this approach many samples of biological origin (biofluids such as urine or plasma) are analyzed using techniques that produce simultaneous detection. A variety of analytical metabolic profiling tools are used routinely, are also currently under development, and include proton nuclear magnetic resonance spectroscopy and mass spectrometry with a prior online separation step such as high-performance liquid chromatography, ultra-performance liquid chromatography, or gas chromatography. Data generated by these analytical techniques are often combined with multivariate data analysis, i.e., pattern recognition, for respectively generating and interpreting the metabolic profiles of the investigated samples. Metabonomics has gained great prominence in diabetes research within the last few years and has already been applied to understand the metabolism in a range of animal models and, more recently, attempts have been done to process complex metabolic data sets from clinical studies. A future hope for the metabonomic approach is the identification of biomarkers that are able to highlight individuals likely to suffer from diabetes and enable early diagnosis of the disease or the identification of those at risk. This review summarizes the technologies currently being used in metabonomics, as well as the studies reported related to diabetes prior to a description of the general objective of the research plan of the metabonomics part of the European Union project, Molecular Phenotyping to Accelerate Genomic Epidemiology.

The use of recently described ionisation techniques for the rapid analysis of some common drugs and samples of biological origin

Three ionisation techniques that require no sample preparation or extraction prior to mass analysis have been used for the rapid analysis of pharmaceutical tablets and ointments. These methods were (i) the novel direct analysis in real time (DART), (ii) desorption electrospray ionisation (DESI), and (iii) desorption atmospheric pressure chemical ionisation (DAPCI). The performance of the three techniques was investigated for a number of common drugs. Significant differences between these approaches were observed. For compounds of moderate to low polarity DAPCI produced more effective ionisation. Accurate DESI and DAPCI tandem mass spectra were obtained and these greatly enhance the selectivity and information content of the experiment. The detection from human skin of the active ingredients from ointments is reported together with the detection of ibuprofen metabolites in human urine.

Csaba Horváth – Scholar, Scientist, Mentor, Friend

At Farbwerke Hoechst, Csaba did research and development work on the surface chemistry of organic pigment dyes and learned about the practical aspects of surface chemistry. During his doctoral research, Csaba invented and developed porous-layer open tubular columns which offers remarkable advantages over wall-coated OTC, e.g., a higher loadability and the ability to use all retention mechanisms afforded by adsorption. He also prepared surface-treated beads, an approach that he used later in the context of HPLC. Working with S.R. Lipsky on the development of analytical methodologies for lunar samples, searching for trace compounds which could show the presence of life on the moon in a distant past, he imagined applying to LC the same principles that he had used earlier in GC, and built the first instrument for high pressure liquid chromatography. Very early, he understood the potential of this new separation method to revolutionize biochemistry and molecular biology. Working for Picker-Nuclear, Csaba developed the first commercial instrument for HPLC, which was also the first instrument to use microbore HPLC columns (for ion-exchange separations of biological compounds). From the beginning, Csaba focused his interests on the separation of samples of biological origin, becoming the pioneer of modern bioanalytical chemistry. Hemore » devoted considerable attention to the development of the theory and applications of reversed-phase liquid chromatography, the most widely applied chromatographic method of analysis, pioneered the use of displacement chromatography for preparative HPLC, and innumerable applications of HPLC to the separation of samples of biological origin. He developed the solvophobic theory of retention in RPLC, the use of entropy-enthalpy compensation in the study of retention mechanisms, and the fundamentals of electrochromatography. The importance of his spearheading HPLC, RPLC, and their applications in the life sciences, fields in which these new methods are leading to countless breakthroughs, is such that his contributions will remain among the major scientific achievemetns of the 20th century.« less

Recent Advances in Analytical Techniques used for the Determination of Fluoroquinolones in Pharmaceuticals and Samples of Biological Origin - A Review Article

Recent Advances in Analytical Techniques used for the Determination of Fluoroquinolones in Pharmaceuticals and Samples of Biological Origin - A Review Article

Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry

Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.

Imaging with a Terahertz quantum cascade laser

We demonstrate bio-medical imaging using a Terahertz quantum cascade laser. This new optoelectronic source of coherent Terahertz radiation allows building a compact imaging system with a large dynamic range and high spatial resolution. We obtain images of a rat brain section at 3.4 THz. Distinct regions of brain tissue rich in fat, proteins, and fluid-filled cavities are resolved showing the high contrast of Terahertz radiation for biological tissue. These results suggest that continuous-wave Terahertz imaging with a carefully chosen wavelength can provide valuable data on samples of biological origin; these data appear complementary to those obtained from white-light images.

Perchloric Acid Enhances Sensitivity and Reproducibility of the Ferric–Xylenol Orange Peroxide Assay

The ferrous oxidation in xylenol orange (FOX) assay for hydroperoxides suffers from very narrow pH optimum in the range 1.7–1.8. Most published protocols recommend 25 mM sulfuric acid as the solvent, but this in practice does not ensure the maintenance of correct pH in the presence of materials such as samples of biological origin. Substitution of perchloric for the sulfuric acid resulted in a lowering of the optimum pH of the assay to 1.1, a decreased dependence of the absorbance of the ferric–xylenol orange complex on acid concentration and decreased sensitivity to added compounds. Molar absorption coefficients of hydrogen peroxide, cumene, and butyl hydroperoxides and of hydroperoxide groups generated in oxidized protein and lipids were determined and found to be higher than in sulfuric acid. The optimum concentration of perchloric acid proved to be 110 mM. The new assay was designated as PCA-FOX, to distinguish it from the FOX methods based on sulfuric acid.

Isolation and Purification Techniques for Pesticide Residue Analyses in Samples of Plant or Animal Origin

Chromatographic methods of pesticide residue analysis of samples of plant or animal origin require isolation of the analyte from the matrix and purification of the crude extract. Combined isolation and clean-up methods are used for these purposes. This paper concentrates on the choice of optimum isolation and purification techniques, which are used for pesticide residue analysis in samples of biological origin.

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C 4, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm 2. The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day ( n=6) and inter-day calibration ( n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.